1521-0111/90/3/341–357$25.00 http://dx.doi.org/10.1124/mol.116.103861MOLECULAR PHARMACOLOGY Mol Pharmacol 90:341–357, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

MINIREVIEW—A LATIN AMERICAN PERSPECTIVE ON ION CHANNELS

Modulation of Ionic Channels and Insulin Secretion by Drugs andHormones in Pancreatic Beta Cells

Myrian Velasco, Carlos Manlio Díaz-García,1 Carlos Larqué, and Marcia HiriartDepartment of Neurodevelopment and Physiology, Neuroscience Division, Instituto de Fisiología Celular, Universidad NacionalAutónoma de México, Mexico City, Mexico

Received February 17, 2016; accepted July 18, 2016

ABSTRACTPancreatic beta cells, unique cells that secrete insulin in re-sponse to an increase in glucose levels, play a significant rolein glucose homeostasis. Glucose-stimulated insulin secretion(GSIS) in pancreatic beta cells has been extensively explored. Inthis mechanism, glucose enters the cells and subsequentlythe metabolic cycle. During this process, the ATP/ADP ratioincreases, leading to ATP-sensitive potassium (KATP) channelclosure, which initiates depolarization that is also dependent onthe activity of TRP nonselective ion channels. Depolarizationleads to the opening of voltage-gated Na1 channels (Nav)and subsequently voltage-dependent Ca21 channels (Cav).

The increase in intracellular Ca21 triggers the exocytosis ofinsulin-containing vesicles. Thus, electrical activity of pancreaticbeta cells plays a central role in GSIS. Moreover, many growthfactors, incretins, neurotransmitters, and hormones can modu-late GSIS, and the channels that participate in GSIS are highlyregulated. In this review, we focus on the principal ionic channels(KATP, Nav, and Cav channels) involved in GSIS and how classicand new proteins, hormones, and drugs regulate it. Moreover,we also discuss advances on how metabolic disorders such asmetabolic syndrome and diabetes mellitus change channelactivity leading to changes in insulin secretion.

IntroductionPancreatic beta cells, which act as glucose sensors, secrete

insulin in response to elevated blood glucose levels. Insulin isan anabolic hormone that regulates the storage of nutrients inthe liver, muscle, and adipose tissue and regulates the glucoseuptake in muscle and adipose tissue. In this way, beta-cellglucose sensing and response are essential for life inmammals(Hiriart et al., 2014).Several nutrients stimulate insulin secretion, although they

must be metabolized, and glucose is the most potent and best-studied secretagogue (“glucose-stimulated insulin secretion,”

GSIS). GSIS involves the interaction between metabolicevents and ion channels activity. Beta cells also expressdifferent types of receptors that modulate GSIS through theiractivation by hormones, growth factors, neurotransmitters,incretins, and drugs (Hiriart et al., 2014).Since 1968, Dean and Matthews (1968) demonstrated that

high extracellular glucose depolarized the membrane ofpancreatic beta cells and increased the action potential firingfrequency in mouse islet beta cells. The activity of at least sixdifferent channels generated the electrical activity in rodentpancreatic beta cells: 1) ATP-sensitive potassium channels(KATP), which determine the resting potential and link glucosemetabolism to electrical activity; 2) transient receptor poten-tial channels (TRPs), which generate nonselective cationiccurrents; 3) voltage-gated sodium channels (Nav); 4) low- andhigh-voltage-activated calcium channels (Cav) that triggeraction potentials to raise cytoplasmic calcium concentrationand insulin secretion; 5) voltage-dependent potassium channels

This work was supported by Dirección General de Asuntos del PersonalAcadémico (DGAPA)-UNAM [Grant IN-213114] to Marcia Hiriart; [GrantIN-211416] to Myrian Velasco; and Carlos Larqué had a CONACyT doctoralfellowship.

1Current affiliation: Department of Neurobiology, Harvard Medical School,Boston, MA.

dx.doi.org/10.1124/mol.116.103861.

ABBREVIATIONS: ABC, ATP binding-cassette; AMPK, AMP-activated protein kinase; CaMKII, calmodulin-dependent kinase II; Cav, voltage-dependent Ca21 channel; CHI, congenital hyperinsulinism; ER, endoplasmic reticulum; FFA, free fatty acid; GLP-1, glucagon-like peptide; GLUT1,Type-1 glucose transporter; GSIS, glucose stimulation insulin secretion; HNF, hepatocyte nuclear factor; KATP channels, ATP-sensitive potassiumchannels; Kv, voltage-dependent potassium channels; LC-CoA, long-chain acyl-CoA esters; MS, metabolic syndrome; Nav, voltage-gated Na1

channels; NBD, nucleotide-binding domain; NDM, neonatal diabetes mellitus; NGF, nerve growth factor; PIP2, phosphatidylinositol-4,5-biphosphate; SNARE, soluble N-ethylmaleimide sensitive factor attachment proteins receptor; SU, sulfonylurea; SUR1, Type 1 sulfonylureareceptor; Syn-1A, syntaxin-1A; T2DM, type 2 diabetes mellitus; TMD, transmembrane domain; TNDM, transient neonatal diabetes mellitus; TRPchannel, transitory receptor potential channel; TTX, tetrodotoxin; VDCC, voltage dependent calcium channels; VGSC, voltage-gated sodiumchannel.

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(Kv); and 6) calcium-sensitive voltage-dependent potassiumchannels that repolarize the membrane potential stoppinginsulin secretion.Beta cells from different mammals express specific combi-

nations of channels (Hiriart and Aguilar-Bryan, 2008; Drewset al., 2010; Hiriart et al., 2014). Despite the fact that thepotential role of each type of ionic channel on insulin secretionhas been discussed in the literature, there is agreement thatthey all participate in this process. Thus, in this review, wewill focus only on extensively studied channels, such as KATP,Nav, and Cav. We examine recent information about thestructure- function relationship of the channels, their role ininsulin secretion, and their regulation by hormones, drugs,enzymes, receptors, and exocytotic proteins. Furthermore, wewill consider some mutations localized in critical structuraldeterminants and how they could impair channel gating, drugbinding, or translocation to the membrane, which couldcontribute to different pathologies.

Glucose-Stimulated Insulin SecretionThe coupling between the rise in extracellular glucose

concentration and insulin secretion involves metabolic andionic events (Hiriart and Aguilar-Bryan, 2008). In the fastingstate, the plasma glucose level is 4–5 mM and beta cells areelectrically silent at their resting potential. Metabolic eventsstart when glucose levels increase above 7mM. Glucose entersthe cells through glucose transporters, GLUT2 (Km 5 11 mM)in rodents or Type-1 glucose transporter (Km 5 6 mM) inhumans (McCulloch et al., 2011). Glucose is then phosphory-lated to glucose-6 phosphate by glucokinase, an essential stepin glycolysis. Phosphorylated glucose enters the glycolyticpathway in the cytoplasm, the Krebs cycle, and oxidativephosphorylation in the mitochondria leading to a rise inintracellular ATP/ADP ratio (Hiriart and Aguilar-Bryan,2008; Drews et al., 2010; Hiriart et al., 2014).ATP inhibits KATP channel activity and reduces K1 efflux.

In this condition, the basal activity of TRP nonselectivecationic channels produces a slow depolarization until athreshold membrane potential is reached that increases theopen probability of Na1 and T-type calcium channel, whichfurther depolarizes the membrane. When the membranepotential reaches approximately 220 mV, high-voltage-activated calcium channels such as P/Q-type, N-type, andL-type calcium channels increase their conductance and raiseintracellular calcium levels, leading to a fast depolarizationand firing of superimposed action potentials. This triggers theexocytosis of insulin-containing granules (Hiriart andAguilar-Bryan, 2008; Drews et al., 2010).Finally, the activity of the delayed rectifier voltage-

dependent potassium channels (Kv) and calcium-sensitivevoltage-dependent potassium channels repolarize the mem-brane, acting as a brake for GSIS (Yang et al., 2014a).High glucose concentration increases the cytosolic Ca21,

ATP, cAMP levels and triggers insulin secretion in a pulsatilemanner, synchronized with calcium influxes during twophases (Jewell et al., 2010). The first phase occurs during 5–10 minutes after beta-cell stimulation and involves the exo-cytosis of plasma membrane-predocked granules, termed thereadily releasable pool. The second phase is less robust and issustained until the glucose stimulation stops. It has beenproposed that it may involve the release of a deeper granule

pool from within the cell named granule storage pool, whichpresumably replenishes the readily releasable pool (Barget al., 2002; Jewell et al., 2010).The molecular mechanism of exocytosis is partly under-

stood and is mediated by soluble N-ethylmaleimide-sensiblefactor attachment protein receptors (SNAREs). SNARE pro-teins such as sintaxyn-1A and SNAP25/23 are located at thecell membrane, whereas vesicle-associated membrane pro-tein or synaptobrevin is anchored to the granule membrane.Interaction among SNARE proteins and other regulatoryproteins such as synaptotagmin, Munc 18-1, Munc 13-1, andGTPase and Rab3A allows the docking, tethering, priming,and fusion to the membrane of insulin-containing granules(Kasai et al., 2010).

ATP-sensitive Potassium ChannelsATP-sensitive potassium KATP channels are considered to

be ametabolic sensor because they couple themetabolic statusof the cell to the membrane potential and its electrical activity(Seino, 2003). KATP channels exist in several tissues, includingbrain, pancreas, and smooth and skeletal muscles, and theirphysiologic role has been mostly characterized in pancreaticbeta cells. As mentioned above, the resting potential in betacells is principally determined by KATP channels (Clark andProks, 2010). In the absence of glucose, beta cells areelectrically silent because the KATP activity is high (3 nS)and K1 efflux is maintained. In this condition, voltage-dependent channels are closed, and insulin secretion occursonly at basal levels. When plasma glucose levels increaseabove 6 mM, KATP channel activity is reduced by more than75%, allowing the depolarization of the cell and increasinginsulin secretion (Rorsman et al., 2011, 2014).

Molecular Structure of KATP Channel and SUR1 NucleotideBinding Domains

The molecular structure of KATP channels consists of hetero-octameric complexes formed by four pore-forming inwardrectifier potassium channel (Kir6.x) subunits and four sulfo-nylurea receptor (SURx) subunits (Aguilar-Bryan et al., 1995).There are two isoforms of inward rectifier potassium channelKir6.1 and Kir6.2 (Clement et al., 1997). The sulfonylureareceptor is an enzymatic member of the ABC (ATP-bindingcassette) superfamily, which uses the energy of ATP bind-ing and hydrolysis to transport substrates across the cellmembrane. This receptor has two nucleotide binding sites(Wilkens, 2015).There are three isoforms of the sulfonylurea receptor:

SUR1, SUR2A, and SUR2B (Devaraneni et al., 2015). Thesulfonylurea receptor is a regulatory subunit because it: 1)confers sensitivity to Mg-nucleotides, 2) is activated by K1

channel openers such as diazoxide and pinacidil, and 3)is inhibited by sulfonylureas such as glibenclamide andtolbutamide (Aguilar-Bryan and Bryan, 1999). In addi-tion, SUR1 increases the open probability of Kir6.2 viaphosphatidylinositol-4, 5-biphosphate (PIP2) (Song andAshcroft, 2001) and may increase the sensitivity to ATP(Tucker et al., 1997).The combination of Kir6.2 (encoded by KCNJ11) and SUR1

(encoded by ABCC8) subunits form the KATP channel in betacells (Fig. 1), and this combination is also present in alpha and

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delta cells from the islet (Clark and Proks, 2010; Ashcroft andRorsman, 2013). Kir6.2 is a member of the inward rectifierpotassium channels superfamily formed by two transmem-brane domains, M1 and M2. These domains are linked by asequence of amino acids that partially enters themembrane toform the pore (loop P) and a signature sequence of glycine-phenylalanine-glycine that corresponds to the K1 selectivityfilter (see Fig. 1) (Xie et al., 2007). Furthermore, the N and Ctermini are intracellular in Kir6.2 channels and are structur-ally important in the regulation by ATP and sulfonylureas.The SUR1 receptor is formed by two transmembrane domains

(TMD1 and TMD2), each one with six transmembrane helices,and two cytosolic H (NBD1 and NBD2) subunits between

the TMDs (Aguilar-Bryan and Bryan, 1999). Moreover, theN-terminal domain (TMD0) has five transmembrane helicesconnected to the TMD1 through a long cytosolic loop known asthe L0 or CL3 linker (Fig. 1) (Devaraneni et al., 2015).Both NBDs have a highly conserved structure, and each one

is formed by a catalytic and an a-helical subdomain. Thecatalytic subdomain contains Walker A and B conservedmotifs that play important roles in ATP binding and hydro-lysis. The A motif (P-loop) contains a highly conserved lysineresidue that interacts with the b and g phosphates of thenucleotides (Devaraneni et al., 2015).The B motif contains an aspartate residue that coordinates

Mg21 binding (ter Beek et al., 2014) and a basic glutamate

Fig. 1. Pancreatic KATP channel structure: (A) Membrane topology of Kir6.2 channel and sulfonylurea receptor (SUR1) subunits. Functional KATP(right) is a 4:4 octamer. Transmembrane domains (TMD0, TMD1, and TMD2): Residues marked in red are associated with neonatal diabetes mellitus(NDM) and those marked in purple are associated with congenital hyperinsulinism (CHI). Retention motif (RKR) is marked in brown. (B)Cooperativity between nucleotide binding domains (NBD1 and NBD2) to form ATP-binding sites, walker A and B motif, loop of glutamine (loop-Q),histidine (loop-H) and aspartate (loop-D). NBS1 binds ATP or MgATP and NBS2 binds MgADP or MgATP. ABC signature sequence (LSGGQ): (C)Pharmacologic modulators of pancreatic KATP channels.

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residue that binds the g-phosphate of ATP through a watermolecule (Beis, 2015). Downstream the Walker B motif, thecatalytic base consists on a glutamate residue that bothhydrolyzes ATP and forms a dyad with the H-loop histidineof the antiparallel NBD (Jones and George, 2012). In addition,the catalytic domain contains functionally relevant residues,such as aspartate (D-loop), that allow the formation of thehydrolytic-competent state and enable the ATP hydrolysis.The a-helical domain contains both the ABC signature

sequence (LSGGQ), also known as C-loop, and a Q-loop(Beis, 2015). Both NBDs are grouped in an antiparallelmanner to form two ATP binding sites (NBS). Thus, WalkerA and B of NBD1 and the signature sequence of NBD2 formthe first ATP-binding site, where nucleotides are located atthe central part of the sandwich (Fig. 1). While in the secondbinding site, A and B motifs of NBD2 are associated with thesignature sequence of NBD1(Vedovato et al., 2015).

Pharmacology of KATP Channels in Pancreatic Beta Cells

Nucleotide Regulation of KATP Channels. The hall-mark of KATP channels is the fine-tuned regulation by in-tracellular ATP that binds to the Kir6.2 channel and triggersits closure. Inversely, the Mg-ADP or Mg-ATP binding to theSUR1 promotes the opening of the KATP channels. Theinhibition by ATP is the most predominant characteristic ofKATP channels. The administration of ATP, with or withoutMg21, inhibits the KATP channels in excised patches with ahalf maximally inhibiting concentration (IC50) in the range of5 to 25 mM (Tarasov et al., 2006; Schulze et al., 2007).Interestingly the intracellular ATP concentration is in amillimolar range (3–5 mM). This observation suggests thatin intact cells, KATP channels should always be closed.However, the fact that KATP channels are open during the

resting potential could be due to several possible reasons:i) cytosolic proteins that regulate the ATP-sensitivity and theKATP channels are loosely bound in inside-out patches; ii) it ispossible that ATP decreases with the activity of membrane-bound ATPases (like Ca21/K1 or Na1/K1), building up an ATPgradient from below the membrane to the cytosol; iii) also,several studies have shown that MgADP opens KATP channelspreviously inhibited by ATP in inside-out patches, suggestingthat the ATP/ADP ratio is more relevant than ATP alone toregulate the KATP channels (Aguilar-Bryan and Bryan, 1999;Drews et al., 2010). Furthermore, the ATP has a greateraffinity to Mg21 than to other cations; therefore, the ATPwithin the cells is attached to Mg21 (Wilson and Chin, 1991).The ATP-inhibition of KATP channels occur through the Kir6.2channel. Experimental data have shown that a truncatedversion of Kir6.2DC36 reaches the plasma membrane andlacks SUR1 interaction. In addition, these channels areinhibited by ATP, but with a lower affinity (Tucker et al.,1997).The ATP binding is mediated by some residues such as the

Arg50 in the N-terminal domain and the Lys185, Arg192,Arg201, and Gly334 in the carboxyl-terminal domain. Thus,both the N- and C-terminal domains of the Kir6.2 channel areinvolved in the ATP binding (Matsuo et al., 2005). Moreover,mutations at Arg192 and Arg201 decrease the sensitivity toATP, ADP, and AMP, suggesting that they interact with theircommon a phosphate. On the other hand, mutations at Lys185specifically diminish the sensitivity to ATP and ADP, due to

an impaired interaction with their common b phosphate(Riedel and Light, 2005). The KATP channels have four ATP-binding sites, one in each Kir6.2 monomer; however, only oneATP molecule is sufficient to inhibit the channel (Markworthet al., 2000).ATP hydrolysis is the canonical property of ABC proteins.

The catalytic cycle starts with the “apo” ground state followedby the binding interaction of NBDs with the TMDs. Thebinding of each NBD to MgATP molecules allow their di-merization and a conformational change of the transmem-brane domains. Finally, the ATP hydrolysis produces thedissociation of NBDs resetting the transporter to the groundstate to start a new cycle (Wilkens, 2015).The ATPase activity of SUR1 drives discrete conformational

changes that modulate the KATP channels gating. Thus, thecatalytic cycle of SUR1 in a prehydrolytic state favors thechannel closing, whereas the posthydrolytic state promotesthe channel opening (Zingman et al., 2001).The SUR1 receptor contains a consensus (NBS2) and a

degenerated (NBS1) ATP binding site. NBS1 binds ATP, butdoes not hydrolyze it (Inagaki et al., 1995; Matsuo et al.,2005). Although the KATP channels are inhibited by MgATPbinding to Kir6.2 subunits, the channels are also activatedwhen MgATP binds to SUR1 (Gribble et al., 1998a). TheMgATP and MgADP interaction with NBDs of the SUR1subunits results in the activation of KATP channels, anincrease of the K1 efflux, and a diminished excitability ofbeta cells. This phenomenon occurs by cooperativity betweenNBD1 and NBD2 (Yang et al., 2014a).Recently, the usage of a heterodimeric ABC protein

(TM287/288) similar to SUR1 suggested that the cooperativitybetween NBDs may be mediated via two D-loops in the dimerinterface. The D-loop at NBS1 ties the NBDs together even inthe absence of nucleotides, while the D-loop inNBS2 is flexible(Hohl et al., 2014). ATP hydrolysis is necessary to switchSUR1 into a conformation that antagonizes nucleotide bindingto the Kir6.2 pore and stimulates channel opening.The ATP hydrolysis occurs in NBD1, which is a high-affinity

site for ATP or MgATP, whereas NBD2 has low-affinity toMgATP orMgADP and intrinsic ATPase activity (Drews et al.,2010; Yang et al., 2014a). However, it has been demonstratedthat MgATP does not activate the channel directly, but ratherit must first be hydrolyzed to MgADP (Zingman et al., 2001).However, recent work demonstrated that nonhydrolyzableATP analogs such as MgAMP-PNP [adenosine 59-(b, g-imino)triphosphate] andMgAMP-PCP [adenosine 59-(b, g,methylene-triphosphate)] fail to activate KATP channels because of selec-tive binding to NBD1 that prevents NBD dimerization and theSUR1 conformational switching, rather than by enzymaticactivity (Ortiz et al., 2013).Regulation by Sulfonylureas. Sulfonylureas are a group

of drugs that decrease blood glucose levels, and for a long time,these compounds were the main treatment of type 2 diabetesmellitus. The sulfonylureas (SUs) were discovered serendip-itously in the 1940s when hypoglycemia symptoms were ob-served after sulfonamide antibiotic treatment (Loubatieres,1957). The first generation of SUs, including tolbutamide,acetohexamide, and chlorpropamide, was used in the 1960s(Deacon and Lebovitz, 2015). The second generation of SUs suchas glibenclamide (glyburide), glipizide, gliclazide, and glimepirideappeared 10 years later. These drugs were produced byreplacing an aliphatic side chainwith a cyclohexyl group, which

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increases its molecular complexity andmolecular affinity to theSUR1 subunit. With this changes, their adverse effects profilewas also improved (Deacon and Lebovitz, 2015).Sulfonylureas were used as an oral antidiabetic for 50 years,

but their action mechanism was unknown. In the 1980, longbefore the cloning of SUR1 in 1995, the high-affinity sites forsulfonylureas were localized in the membrane of beta cells(Gaines et al., 1988; Aguilar-Bryan et al., 1995). According tothe previously described model for the SUs and SUR in-teraction, there are two binding sites located on the eighthcytosolic loop between TM segments 15 and 16 and the thirdcytosolic loop between TM segments 5 and 6 (Ashfield et al.,1999), and it has been proposed that glibenclamide andglimepiride bind to both sites in the SUR1 receptor(Mikhailov et al., 2001), and recently suggested that theKir6.2 N-terminus participates in this binding (Vila-Carrileset al., 2007; Kühner et al., 2012). On the other hand,tolbutamide binds only to the high-affinity site of SUR1 inthe intracellular loop between TM 15 and 16. The substitutionof tyrosine for serine 1237, localized between TM 15 and 16,blunts the channel inhibition by tolbutamide (Ashfield et al.,1999). Furthermore, it was demonstrated that tolbutamidealso binds to a low-affinity site in the Kir6.2 channel (Gribbleet al., 1997). SUs bind with different affinity to the threeisoforms of SUR receptor (SUR1, SUR2A, and SUR2B), and,interestingly, the SUR1 receptor that forms the pancreaticKATP channels has the highest affinity to sulfonylureas with adissociation constant in the nanomolar range (Aguilar-Bryanand Bryan, 1999).KATP channels increase their opening time in response to

several compounds known as potassium channel openers (seeFig. 1). The principal potassium channel openers are pinacidil,cromakalin, and diazoxide; the SUR1 receptor responds betterto diazoxide than SUR2A cardiac receptor, but they do notrespond to pinacidil (Aguilar-Bryan and Bryan, 1999).KATP trafficking regulation. Nucleotides are not the

only protein that regulates KATP channels activity; a higherlevel of complexity in the structure-function relationship isadded due to their regulation by other proteins. Particularattention has been paid to proteins that regulate the traffick-ing of KATP channels to the membrane. Biogenesis andassembly of KATP channels occur in the endoplasmic reticulum(ER); however, when Kir6.2 or SUR1 subunits are expressedalone, they are trapped in the ER and degraded, suggestingthat assembled complexes are required for the forwardtrafficking and the surface expression (Zerangue et al.,1999). Both subunits are needed to form a fully functionalKATP channel, and these subunits are assembled in the ER.The assembling occurs through a tripeptide (RKR) retentionmotif within the C terminus of Kir6.2 and between TMD1 andTMD2 of SUR1 (Tucker et al., 1997; Zerangue et al., 1999).In 2004, an interaction between KATP channels and syn-

taxin 1A was demonstrated (Syn-1A; t-SNARE protein). Also,it was proposed that syntaxin 1A (Syn-1A) interacts withNBDs of SUR1 and inhibits the KATP channel activity (Pasyket al., 2004). It has been observed that the levels of Syn-1A andcognate SNARE proteins are reduced in diabetic human androdent beta cells (Nagamatsu et al., 1999; Ostenson et al.,2006). In addition, overexpression and downregulation of Syn-1A led to a decrease and an increase in the surface expressionof KATP channels, respectively. It was suggested that the lowerexpression of KATP channels at the membrane is caused by an

accelerated endocytosis and a decreased biogenesis, as well asaltered traffic to themembrane of these channels. The authorseven proposed that physiologic and pathologic changes in Syn-1A expression may control the KATP channel expression, thusmodulating insulin secretion (Chen et al., 2011).Leptin, a product of the LEP/ob gene, is an obesity-related

hormone, which is predominantly secreted by adipocytes indirect proportion to body fat mass. Leptin inhibits insulinsecretion by increasing the cell-surface expression and theopen probability of KATP channels, resulting in a more hyper-polarized membrane potential (Holz et al., 2013). Leptin alsoinduces an increased surface expression of Kv2.1 channels,leading to a rapid repolarization of membrane potential (Parket al., 2013b). These studies suggest that leptin exerts acoordinated trafficking regulation of KATP and Kv2.1 channelsto inhibit insulin secretion (Wu et al., 2015). Furthermore,leptin activates the AMP-activated protein kinase (AMPK)through phosphorylation by the Ca21/calmodulin-dependentprotein kinase kinase beta and increases KATP channelstrafficking (Park et al., 2013a).The translocation of KATP channels to the plasma mem-

brane could be promoted byAMPKactivation, which regulatesthe actin cytoskeleton through the activation of Rac GTPaseand phosphorylation of myosin regulatory light chain inpancreatic beta cells. The trafficking of K1 channels to cellsurface could also be considered as a novel autocrine negativefeedbackmechanism for insulin secretion (Han et al., 2015). Inthis way in MIN6 cells, high glucose and exogenous insulintreatment increase KATP channels trafficking to the surface,stabilizing the membrane potential and avoiding insulinsecretion. This increase in cell-surface KATP channels ismediated by vesicle-associated membrane protein 2 via thephosphoinositide 3 kinase (Xu et al., 2015). The hormonal andmetabolic regulation of KATP channel trafficking constitutesan emerging field of potentially high significance to beta-cellphysiology.

Pancreatic KATP channelopathies

Functional and structural defects in KATP channels impairinsulin secretion, leading to the onset of diseases. In 1981, itwas first described that glucose failed to promote insulinsecretion in a concentration-dependent manner in isolatedtissue from a child with hyperinsulinemia-induced hypogly-cemia (Aynsley-Green, 1981; Dunne, 2000). The persistenthyperinsulinemia hypoglycemia of infancy, also named con-genital hyperinsulinism (CHI), is a complex disorder com-posed of clinical, morphologic, and genetic changes (Shahet al., 2014). Excessive insulin secretion characterizes CHI,despite low blood glucose levels in the early neonatal period.This disorder has an incidence of around 1 in 50,000 livebirths.Clinically, CHI is classified in mild and severe forms of the

disease. The first one appears during the newborn period, butalso in infancy and childhood. In the severe form thesymptoms develop after the first hours or days after birth.Failure in the diagnosis and treatment of hypoglycemia couldlead to severe brain damage and epilepsy (Kapoor et al., 2009).Histologically, CHI is divided into diffuse, focal, and

atypical forms. The focal form is sporadic in inheritance andonly affects discrete areas of the pancreas. The diffuse diseaseis inherited in an autosomal recessive or dominant way. The

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latter is the most common of CHI and affects the entirepancreas. Finally, the atypical forms may be diffuse along thegland with normal or abnormal islet anatomy (Shah et al.,2014).The etiology of CHI may be due to rare variants in nine

different genes, such as: 1) ABCC8 (SUR1 receptor), 2) KCNJ11(Kir6.2 channel), 3) GCK (glucokinase), 4) GLUD1 (mito-chondrial enzyme glutamate dehydrogenase), 5) SLC16A1[monocarboxylate transporter 1 (MCT1)], 6) HADH (mito-chondrial L-3-hydroxyacyl-coenzyme A dehydrogenase), 7)UCP2 (uncoupling protein 2), and 8 and 9) HNF4A andHNF1A(hepatocyte nuclear factor 4alpha and 1alpha). All of thesegenes encode for proteins involved in beta-cell insulin secre-tion (Ro�zenková et al., 2015). The most frequent causes forCHI are variants in KCNJ11 and ABCC8, which accountfor∼70% of all cases withmore than 200mutations reported todate, some of them shown in Fig. 1. Both are inherited either byautosomal recessive or dominant form (Remedi and Koster, 2010).KATP channel variants can also be grouped into two

functional classes: those that reduce: i) the cellular membraneexpression by affecting either channel biosynthesis, traffick-ing, assembly; or ii) reduce the intrinsic channel activity(Remedi and Koster, 2010). Genetic variants in ABCC8 andKCNJ11 along with HNF1A and GCK are associated notonly with CHI but also with maturity onset of diabetes inyoung and neonatal diabetes mellitus (NDM), each of themare monogenic forms of glucose-regulation disorders in children.Neonatal diabetes mellitus (NDM), caused either by Kir6.2

(KCNJ11) or SUR1 (ABCC8) mutations, is a rare disorderthat appears during the first 6–9 months of life, with anincidence of 1:300,000 live births. The patients typically havelow weight at birth, and only one-third of them develop severeketoacidosis. NDM can be permanent or transient (TNDM)and requires lifetime glycemic control or until a remissionperiod. Moreover, some patients present amore severe form ofNDM with severe developmental delay and epilepsy, whereasother patients show a milder phenotype without epilepsy.NDM-associated KATP channel mutations cause the oppo-

site phenotype of CHI. Therefore, they have an overactivechannel that remains opened, either increasing the KATP

activity mediated by Mg nucleotides or altering the intrinsicgating (Edghill et al., 2010; Remedi and Koster, 2010).Gloyn et al. (2004) reported genetic variants in Kir6.2 as the

potential cause of NDM for the first time in 2004. To date, over30 rare variants in Kir6.2 and approximately 15 in SUR1 havebeen identified and associated with NDM. These are the mostcommon cause of permanent NDM (approximately 30–50%)and involved in 15% of the TNDM cases (Shimomura, 2009;Shimomura et al., 2009). TNDM also correlates with muta-tions in hepatocyte nuclear factor-1beta (HNF1b), which canalso cause maturity-onset diabetes of the young 5. Further-more, the majority of cases of TNDM are due to abnormalitiesof an imprinted locus on chromosome 6q24 that results in theoverexpression of a paternally expressed gene (Aguilar-Bryanand Bryan, 2008).Electrophysiological studies have observed that Kir6.2

variants at Arg50, Ile192, Arg201, and Phe333 residuesinvolved in ATP binding decrease the KATP sensitivity.In addition, variants of Val59, Cys166, Ile197, and Ile296residues involved in the channel gating impair the openprobability of the channel (Proks et al., 2005; Edghill et al.,2010).

SUR1 mutations affect either the Mg-nucleotide-mediatedactivation or the intrinsic gating. These mutations could belocated along the protein length, including many instances ofthe following amino acid substitutions V1523A, V1524M,I1425V, and R1531A in NBD2 (Edghill et al., 2010).Mutations and polymorphism in Kir6.2 (KCNJ11) have

already been linked to type 2 diabetes mellitus (Gloyn et al.,2003). Several genetic studies consistently demonstrated anassociation of the E23K polymorphism with a reduced insulinsecretion in glucose-tolerant and -intolerant cohorts as a riskallele in the development of type 2 diabetes (Nielsen et al.,2003; Chistiakov et al., 2009; Villareal et al., 2009).The E23K polymorphism is due to a substitution of a

glutamic acid (E) by a lysine (K) at codon 23 of the KCNJ11.This residue substitution is located in the N-terminal domainof the Kir6.2 subunit (Bonfanti et al., 2015).Electrophysiological recording of reconstituted KATP chan-

nels in mammalian expression consistently shows hyperac-tivity of E23K channels, which has been suggested to increasethe intrinsic channel open probability with a decrease in thesensitivity to ATP inhibition (Villareal et al., 2009; Remediand Koster, 2010) or enhanced activation by long-chain acylcoenzyme A (CoA) esters with minimal effects on ATPsensitivity (Riedel and Light, 2005).Impairment of the KATP channel activity can alter beta-cell

electrical activity and insulin secretion sufficiently to causediabetes. The Kir6.2-G324R mutation reduces the channelATP sensitivity in beta cells; however, the difference in ATPinhibition between homozygous and heterozygous patientswas remarkably small. Nevertheless, the homozygous patientdeveloped neonatal diabetes, whereas the heterozygous par-ents were unaffected (Vedovato et al., 2016).

Endogenous Regulation of KATP Channels in the Beta Cell

The regulation of KATP channels by endogenous ligandscould be required for the appropriate function under physio-logic conditions. ATP/ADP ratio is the principal physiologicregulator of these channels; however, they can also beregulated by lipids, such as long-chain acyl-CoA esters (LC-CoAs), phosphatidylinositol-4, 5-biphosphate (PIP2) (Tarasovet al., 2004), syntaxin-1A (Syn-1A) (Pasyk et al., 2004), andleptin (Gavello et al., 2016).Acyl CoAs are products of free fatty acid (FFA) esterification

by acyl-CoA synthetase. Larsson et al. (1996) demonstratedthe activation of KATP channels by LC-CoAs at a physiologicconcentration in mice and clonal pancreatic beta cells.LC-CoAs esters are also potent stimulators of KATP channelsactivity in human beta cells and do not require the presence ofMg21 or nucleotides (Bränström et al., 2004). The increase inthe activity of KATP channels occurs by interaction of LC-CoAsin the carboxyl-terminal domain of Kir6.2 subunit (Gribbleet al., 1998b), where the Lys332 residue is a key structuraldeterminant (Bränström et al., 2007). Moreover, the effects ofLC-CoAs on the activity of KATP channels depend on both thelength and the saturation degree of the acyl chain. Saturatedacyl CoAs are the most potent activators of KATP channels,followed by monounsaturated and polyunsaturated acylCoAs, respectively (Riedel and Light, 2005).In addition, the KATP channels with E23K (0.6471 allele

frequency) and I337V (0.6448 allele frequency) Kir6.2 poly-morphisms, which are frequent in Caucasian type 2 diabetes

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populations, showed significantly increased activity in re-sponse to palmitoyl-CoA, compared with wild-type KATP

channels. These observations suggest that the diabetogeniceffects of E23K and I337V polymorphisms occur through anincreased KATP channel activity mediated by high levels ofLC-CoAs (Riedel et al., 2003).To date, there is clear evidence supporting the regulating

role of phosphoinositides in the activity of many ion channels(Hille et al., 2015). Fan and Makielski (1997) showed for thefirst time that the KATP channels are regulated by thephosphatidylinositol 4-5 biphosphate (PIP2). PIP2 is a secondmessenger that increases the activity of KATP channels.Electrostatic interaction occurs between negative phosphatehead groups of PIP2 and positively charged residues in Kir6.2(Schulze et al., 2003), including Arg54 in N terminus andArg176, Arg177, and Arg206 in C terminus. All of theseresidues are localized above the ATP binding site and nearto the plasma membrane (Ribalet et al., 2005).Several studies demonstrated that SUR1 plays an impor-

tant role in stabilizing the interaction of PIP2 with the Kir6.2subunit. Recently, a variant (E128K) in the TMD0 of SUR1showed diminished the open probability and the responses toPIP2 (Pratt et al., 2011), raising the possibility that TMD0 ofSUR1modulates the Kir6.2 gating by reducing the response toPIP2. Thus, PIP2may play an important role in regulating theactivity of K1 channels and, therefore, insulin secretion.Furthermore, PIP2 can interact with other proteins that alsoregulate the KATP channel activity, and these proteins appearto be involved with the exocytotic machinery [e.g., synapto-tagmins, calcium-dependent activator protein for secretion,and syntaxin-1A (Syn-1A)]. Syntaxin-1A is a target protein ofSNARE complex that regulates the KATP channel trafficking(Pasyk et al., 2004) and is a potent inhibitor endogenous ofKATP channels (Chang et al., 2011). PIP2 directly and in-directly regulates the interaction of syntaxin-1A on SUR1.PIP2 also affects the Syn-1A binding on NBDs of SUR1, albeitPIP2 acts indirectly on syntaxin-1A clusters on the plasmamembrane to sequester Syn-1A and reduce its availabilityand interaction with SUR1 at the plasma membrane (Lianget al., 2014).Leptin is an adipocyte-derived hormone that regulates food

intake, body weight, glucose homeostasis (Friedman andHalaas, 1998), and was recently demonstrated as a regulatorof KATP trafficking within beta cells (Holz et al., 2013).Moreover, leptin can activate different ion channels, includingKATP channels, both in central neurons and beta cells (Gavelloet al., 2016). The mechanism by which leptin activates KATP

channels involves phosphatase and tensin homologe inhibi-tion mediated by phosphoinositide 3 kinase. phosphatase andtensin homologe inhibition induces F-actin depolymerizationand, consequently, KATP channel opening (Park et al., 2013b;Wu et al., 2015).

The Role of KATP Channels in the Pathophysiology of MSand T2DM

In obesity and type 2 diabetes, acyl CoAs, PIP2, and Syn-1Alevels change. Chronic plasma levels of FFA in obese (Golayet al., 1987) and diabetic patients (Reaven et al., 1989) canincrease the cytosolic acyl CoAs and KATP activity (Riedelet al., 2003). Long-chain polyunsaturated fatty acids maintaincell membrane fluidity and facilitate the cellular signaling

mechanisms. Long periods of high-fat diet, particularly satu-rated fat and trans fatty acids, alter cell membrane lipidcomposition, compromising transmembrane insulin receptorsignaling and consequently glucose uptake peripheral tissues(Wilcox, 2005). Furthermore, in diabetic human and rodentbeta cells, the levels of Syn-1A and cognate SNARE proteinsare reduced (Nagamatsu et al., 1999; Ostenson et al., 2006).The increase in adipose mass will elevate plasma leptin levelsand this, in turn, will feed back to pancreatic beta cells toinhibit insulin secretion via stimulation of KATP channels(Campfield et al., 1995). This suggests that during pathophys-iological conditions such as obesity, insulin resistance, ormetabolic syndrome, the regulation of KATP channels couldbe affected and may contribute to the disease. Recent studieshave evaluated the KATP activity during the metabolicsyndrome (MS), which is a group of symptoms includingcentral obesity, hyperinsulinemia, insulin resistance, highplasma levels of triglycerides, and high arterial blood pres-sure that predispose the subject to develop type 2 diabetesmellitus, cardiovascular diseases, and certain types of cancer.In contrast to type 2 diabetes mellitus, where there is anirreversibly reduced insulin secretion and pancreatic beta-cell exhaustion, the MS condition may be reversible, allowingthe study of channel modifications before type 2 diabetesmellitus develops. A metabolic syndrome model in Wistarrats, developed by adding 20% sucrose to the drinking water for6 months, was used to study the KATP channel activity andATP sensitivity in inside-out patches (Velasco et al., 2012).During the metabolic syndrome, the ATP sensitivity of theKATP channels is higher than in the control. The dissociationconstant (Kd) and Hill coefficients for ATP interaction withKATP channels were 18.3 6 0.01 and 1 6 0.01 mM for controland 10.16 0.9 and 0.96 0.01 mM for MS cells, respectively. Itwill be important in the future to investigate the mechanismsof this change, considering that the intracellular environ-ment modulates KATP channels activity and eventually theexcess of ATP cause the channel dysfunction in type 2 diabetesmellitus (Velasco et al., 2012).

Voltage-Gated Na1 ChannelsVoltage-gated Na1 channels (VGSC) have not been studied

as exhaustively as other channels in beta cells. However, it iswell known that modulators of these channels can influenceinsulin secretion (Diaz-Garcia et al., 2010), and severalreports suggesting novel roles of Na1 channels in pancreaticbeta cells have appeared. Therefore, we will address the rolesfor these channels in beta- cell physiopathology.

Structural Determinants of VGSC

Voltage-gated Na1 channels (VGSC) are multimeric pro-teins. The core of the channel is formed by the alpha subunit,which encodes four homologous domains (I–IV), each of themcomprising six membrane-spanning alpha helices (S1–S6)(Ahern et al., 2016). Nine members (Nav1.1–1.9 or Scn1–9a)constitute the gene family of VGSC alpha subunits (Catterallet al., 2005). Among them, Nav1.7 and Nav1.3 are widelyexpressed in pancreatic beta cells of rodents (Vignali et al.,2006; Zhang et al., 2014), whereas Nav1.6 and Nav1.7 areexpressed in humans (Braun et al., 2008).

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Alpha subunits share a prototypical architecture (Fig. 2A),where the segments S5–S6 of each domain and the extracel-lular loop that connects them (called P, denoting pore),constitute the hydrophilic pore that allows the movement ofions and determines the particular selectivity of the channelfor Na1. Furthermore, the S4 segments serve as voltagesensors due to positively charged residues in these regions(Catterall, 2000a; Ahern et al., 2016). These channels activatein response to membrane depolarization, but their activitydecreases during a sustained stimulus, a phenomenon knownas inactivation (Fig. 2B) (Ahern et al., 2016). The biophysicalproperties of alpha subunits, as well as their trafficking, areregulated by the interacting VGSC beta subunits, whichcomprise 4 genes and 5 members, as one of them presentstwo splicing variants (O’Malley and Isom, 2015). Among thelatter, the predominant beta subunit expressed in pancreaticbeta cells is Scn1b (Ernst et al., 2009; Zhang et al., 2014).

Historical Overview of Nav Channels and Insulin Secretion

Direct reports on functional VGSC in insulin-secreting cellsfirst appeared in the 1980s, when a transient inward Na1

current was described in RINm5F cells, which could beblocked by 2.5 mg/ml of tetrodotoxin (TTX) (Rorsman andTrube, 1986). This finding was in contrast to a previous reportfrom the same group on native pancreatic beta cells fromNMRI mice, where the authors found no effect of TTX (20 mM)on inward currents elicited by membrane depolarizationusing the whole cell patch clamp recordings (Rorsman andTrube, 1986). At that time, a plausible explanation was that

voltage-activated Na1 currents could reflect a dedifferentiatedstate of RINm5F cells (Rorsman et al., 1986). However, becausetheRINm5Fcell linewas derived froma rat insulinoma, species-related differences could also account for this discrepancy.The report of a TTX-sensitive Na1 current in isolated

pancreatic beta cells (Plant, 1988), helped advance an un-derstanding of previous negative results in beta cells fromNMRI mice. This report determined that the voltage for half-maximal steady-state inactivation (V0.5) was more negativethan 2100 mV at regular (2.6 mM) or low (0.2 mM) Ca21

concentrations in the bath solution. The latter argued againsta potential role of VGSC in insulin secretion in mice, becausethe resting membrane potential is around 270 mV at lowglucose, a potential at which the channels ofmice beta cells areexpected to be fully inactivated. Consistently, the applicationof 1.5 mM TTX did not alter the electrical responses whenglucose rose from 3 to 20 mM (Plant, 1988).Almost at the same time, Hiriart and Matteson (1988)

demonstrated that this was not the case for rat pancreaticbeta cells. They consistently recorded voltage-activated Na1

currents with an almost complete steady-state inactivationaround240 mV, which was rightward shifted, compared withthe study of Plant (1988) in mice. Accordingly, the averageinsulin release of individual beta cells was decreased byincubating with TTX (200 nM), as measured by a reversehemolytic plaque assay at high glucose (15.6 mM) (Hiriart andMatteson, 1988).Studies in other species (such as dogs and humans)

confirmed the expression of VGSC (Philipson et al., 1993), aswell as their contribution to the spiking activity of pancreatic

Fig. 2. Structure and biophysical properties ofvoltage-gated Na+ channels. (A) The cartoonrepresents the structure of the pore-formingalpha subunit. In each domain (D-I to D-IV), S4segments show positive charges and the S5–S6segments with the P-loop in between are high-lighted in red. (B) Schematic representation ofthe closed, open, and inactivated states of VGSC.At hyperpolarized membrane potentials (i.e., theresting membrane potential of 270 mV in pan-creatic beta cells) Nav channels remain in theclosed state. A depolarizing stimulus causes thetransition to an open state in Nav channels,where Na+ ions permeate through the channelpore. A sustained depolarization induces theinactivation of the channel, represented as asecond gate that prevents the flow of ions evenwhen the activation gate is still open. The re-covery from inactivation occurs at hyperpolarizedpotentials. (C) The classic role for Nav channelsin beta-cell physiology involves their activationafter the high glucose-dependent KATP channelclosure, which in turn enhances depolarizationand spiking activity, promoting Ca2+ entry andinsulin secretion. The scheme also summarizessome of the pharmacological manipulations thatpotentiate (right panel, sharp arrowhead) orinhibit (let panel, blunted arrowhead) VGSCactivity and consequently modulate glucose-stimulated insulin release

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beta cells. Similar results have been obtained in porcine betacells (Pressel and Misler, 1990), where TTX (1 mM) blocksvoltage-gated Na1 currents and action potentials elicited by12 mM glucose (Silva et al., 2009). Even in mice, where VGSCswere supposed to marginally participate in beta-cell excitabil-ity because of inactivation at physiologic membrane potential(Plant, 1988), the genetic deletion of the ancillary beta 1 sub-unit (Scn1b) impairs the glucose-induced insulin secretion,causing glucose intolerance (Ernst et al., 2009).Recently, Zhang et al. (2014) examined the role of VGSC in

pancreatic beta cells of mice, combining several pharmacolog-ical and genetic tools and using state-of-the-art models for theassessment of beta-cell physiology (i.e., intact islet electro-physiology, insulin release measurements in isolated islets,and perfused pancreas). The authors found that althoughScn9a/Nav1.7 is the isoform with the highest expression levelin this model, the form Scn3a/Nav1.3 is also expressed andsignificantly contributes to VGSC-mediated currents in 30%of the beta-cell population (Zhang et al., 2014). Accordingly,glucose-induced insulin secretion was impaired in pancreaticislets from Scn3a2/2 but not in Scn9a2/2mice (Zhang et al.,2014). These results support that a minor fraction of cellsexpressing higher relative amounts of Scn3a/Nav1.3 tran-scripts may exhibit greater excitability and mediate therecruitment through gap junction coupling of other beta cellswith less sensitivity to rises in glucose concentration (Zhanget al., 2014).Indeed, VGSCmay be a clinically relevant target, because a

decrease in their activity profoundly impacts beta-cell excit-ability and insulin release in humans. In an early paper,Pressel and Misler (1990) showed that replacing ∼94% of theextracellular Na1 with N-methylglucamine abrogated theaction potentials elicited by 10 mM glucose in human pancre-atic beta cells. Later, it was demonstrated that 1 mM TTXreduced the amplitude and broadened the width of the actionpotential of beta cells from human donors (Barnett et al.,1995). As expected, TTX application (as well as Na1 sub-stitution) in the bath solution significantly reduced thevoltage-activated Na1 currents in these cells, whereas thetoxin impaired insulin secretion when islets were challengedfrom 3 to 6 mM glucose (Barnett et al., 1995). Braun andcoworkers (2008) obtained similar results using TTX, whichreduced insulin secretion in 6 and 20 mM glucose of isolatedpancreatic islets from human donors. These authors alsocorroborated that TTX (0.1 mg/ml) decrease the amplitude ofvoltage spikes in pancreatic beta cells, as well as the voltage-gated Na1 whole cell currents (Braun et al., 2008).The molecular identity of the channels mediating these

currents has also been explored in human pancreatic islets,where the highest mRNA levels corresponded to Nav1.6 andNav1.7 (Braun et al., 2008). However, only aminor componentof the voltage-dependence inactivation of Na1 currents re-sembled that of beta cells of mice with an inactivation V0.5 of2105 mV (Zhang et al., 2014). The hyperpolarizing shift inthe voltage of half-maximal inactivation for Nav1.7 in pancre-atic beta cells seems to occur through the interaction with abeta-cell-specific intracellular factor (Zhang et al., 2014), yet tobe determined. Changes in the activity of Nav1.7 channelsmay be linked to beta-cell dysfunction and the susceptibility ofdeveloping painful neuropathy, because this channel is alsoexpressed in dorsal root ganglion neurons (Hoeijmakers et al.,2014). Recently, it was shown that Nav1.7 channels are

linked to insulin production, because pancreatic islets fromScn9a2/2mice exhibit increased insulin content comparedwiththeir wild-type counterparts (Szabat et al., 2015). The latteralso offers new perspectives for drugs like carbamazepine,which inhibits the channel and dose dependently increasesthe mRNA expression of both insulin genes (Ins1 and Ins 2),as well as Pdx1 (a marker of pancreatic beta cells) in isolatedislets from mice (Szabat et al., 2015).

Pharmacology of Nav Channels in Pancreatic Beta Cells

From the previous section, it is evident that tetrodotoxin, apotent blocker of voltage-gated Na1 channels, stands out asthe first-choice pharmacological agent to use in the study ofthese proteins. Toxins have been extensively used to explorethe structure function of ion channels (Morales-Lazaro et al.,2015) and also because their high potency and selectivity canbe harnessed in a more complex pathophysiological context(Diaz-Garcia et al., 2015). The chemical structure and themechanisms of action of these bioactive molecules are diverse.The toolkit of toxins with proven effects on insulin secretionvia VGSC includes compounds isolated from plants, snakes,and scorpions, which modulate the biophysical properties ofthese channels instead of blocking the pore, as TTX does. Theuse of toxins like veratridine, which prevents channel in-activation (Ulbricht, 2005) through the neurotoxin interactionsite 2 (Cestele and Catterall, 2000), established the presenceof VGSC in pancreatic beta cells years before the first directelectrophysiological recordings. An early paper fromDonatschet al. (1977) showed that veratridine (100 mM) increasesinsulin secretion in isolated islets from albino rats perfusedwith 5.6mMglucose to a similar extent to that observedwith arise in glucose to 16.7 mM. Moreover, these authors demon-strated that the secretagogue activity of veratridine could bereversibly inhibited by 3 mM TTX (Donatsch et al., 1977).The effect of veratridine (200mM) andTTX (3mM) on insulin

secretion was later corroborated by Pace (1979) in pancreaticislets from Sprague-Dawley rats. The author also observed averatridine-induced depolarization of pancreatic beta cells inculture, as well as an increase in their spiking activity (Pace,1979). In agreement with these findings, the ability ofveratridine (30 mM) to induce Na1 oscillations in islets cellsfrom ob/ob mice was prevented by TTX (Grapengiesser, 1996).A concentration of 10 mM veratridine was not as effective as a20-fold higher concentration, but increased insulin secretionwhen 20 nM of Leiurus toxin was applied simultaneously(Pace and Blaustein, 1979). The scorpion toxin from Leiurusquinquestriatus also delays the inactivation of Na1 currents(Gonoi et al., 1984), which is a common property of alpha-scorpion neurotoxins and certain spiders’ toxins interactingwith the neurotoxin receptor site 3 in the alpha subunit ofVGSC (Cestele and Catterall, 2000). Another alpha-scorpionneurotoxin, TsTx-V (5.6 mg/ml) from Tityus serrulatus hasbeen shown to reversibly increase the electrical activity ofpancreatic beta cells from Swiss mice perfused with 11 mMglucose, in a similar fashion as 110 mMveratridine (Goncalveset al., 2003). Moreover, TsTx-V also increases the insulinsecretion of pancreatic islets from Wistar albino rats whenincubated with 2.8 or 8.3 mM glucose. Crotamine, a toxin fromthe rattlesnake Crotalus durissus terrificus, also promotesinsulin release by preventing VGSC inactivation. An activefraction (F2) obtained from reversed-phase high-performance

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liquid chromatograph, enhanced insulin secretion of rat isletsincubated with 16.7 mM glucose, exhibiting a threefoldaugment in potency with respect to the native crotamine(Toyama et al., 2000).The effect of certain drugs has been studied on the late Na1

currents from insulin secreting cells, which increase afterlong-term exposure to 33 mM glucose, a condition thatresembles hyperglycemia (Rizzetto et al., 2015). Notably, theblockers ranolazine (10 mM) and TTX (0.5 mM) decrease thespiking activity of INS-1E cells when stimulated by highglucose or tolbutamide, as well as the tolbutamide-inducedCa21 transients (Rizzetto et al., 2015). Also, both blockersattenuated the potentiating effect of veratridine (40 mM) onglucose-stimulated insulin secretion (Rizzetto et al., 2015).Recently, VGSC were identified as regulators of cytokine-

induced beta-cell death. This finding arose from the discoveryof carbamazepine as a protecting agent against a cocktail ofcytotoxic cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) (Yang et al., 2014b). In thestudy, the authors demonstrated that carbamazepine (0.01–100 mM) dose dependently reduced the cytokine-inducedapoptosis in beta cells and expanded these findings to otherVGSC blockers such as lidocaine and TTX (Yang et al., 2014b).Interestingly, carbamazepine (0.3–60 mM) also prevents thelipotoxicity of palmitate (0.5 mM) on beta cells (Cnop et al.,2014). These results shed new light on unexpected roles forVGSC in inflammatory and metabolic events that may occurin diabetes mellitus.

Nav Channels Modulation by Endogenous Molecules

The activity of VGSC can also be modulated by endogenoussignals, usually acting as downstream effectors of signalingcascades. This phenomenon was observed since the initialstudies onVGSC in insulin-secreting cells, because the chronic(48 hours) application of phorbol ester (TPA, a PKC activator)was shown to decrease the Na1 current density in RINm5Fcells (Rorsman et al., 1986). Nav channel activity may pre-dispose to beta-cell death after an inflammatory insult (Yanget al., 2014b). On the contrary, nerve growth factor (NGF)favors beta-cell survival and insulin secretion (Vidaltamayoet al., 2002; Navarro-Tableros et al., 2004). It has beendemonstrated that rat beta cells exhibited higher Na1 currentdensities when incubated with 50 ng/ml of 2.5S NGF for5 days, which could be prevented by either actinomycin D orcycloheximide (Vidaltamayo et al., 2002). The latter suggestedthat the NGF-mediated increment in Na1 currents may bedue to an increased transcription and protein synthesis, whichis supported by elevated Nav1.3 transcripts in the NGF-treated cells (Vidaltamayo et al., 2002).The ability of NGF to enhance the activity of voltage-gated

ion channels in pancreatic beta cells is not exclusive to theNavfamily, because it has also been demonstrated for Ca21

channels (Navarro-Tableros et al., 2007a). In both cases, theeffects are mediated by TrkA, a high-affinity receptor withintrinsic tyrosine kinase activity (Rosenbaum et al., 2001).Remarkably, the inhibition of protein tyrosine kinases leads toimpaired insulin secretion (Persaud et al., 1999), which agreeswith the role of these signaling molecules in maintainingproper beta-cell excitability.Nav channels may also serve as downstream effectors in

G-coupled receptor cascades. The muscarinic agonist carbachol

(100 nM), for instance, promotes insulin release from isolatedrat beta cells at low glucose concentrations (0–5.6 mM), whichcan be prevented by the coapplication of 319 nM TTX (Hiriartand Ramirez-Medeles, 1993). However, the role of Nav inthe muscarinic signaling, at high glucose (20.6 mM), is notstraightforward. Strikingly, carbachol decreases insulin se-cretion in this condition, as well as its inhibitory effect,and is not additive with that of TTX (Hiriart and Ramirez-Medeles, 1993).A recent work from Salunkhe and coworkers (2015)

demonstrated that the microRNA-375 also modulates theactivity of Nav channels in insulin-secreting cells. Theauthors observed that the overexpression of microRNA-375in INS-1 832/13 cells led to a decrease in the protein levels ofScn3a and Scn3b. Nevertheless, the relevance of this regu-lation is not completely understood in a more physiologiccontext, because glucose-induced insulin secretion was notaffected by knocking down microRNA-375 (Salunkhe et al.,2015).Interestingly, Nav channels are also connected to ATP

levels in beta cells. It was proposed that Na1 currents maybe enhanced by an increase in glucose levels and further ATPproduction in beta cells (Zou et al., 2013). This idea issupported by whole cell recordings of beta cells in slices fromrat pancreas, where concentrations of ATP (2–8 mM) in therecording pipette shift the inactivation curve to more positivepotentials and accelerate the recovery from inactivation ofVGSC (Zou et al., 2013). In the other direction, the TTX-sensitive depolarizing Na1 influx contributes to the cytosolicNa1 transients (Nita et al., 2014), which, together with Ca21

transients, propagate to the mitochondria and modulateATP synthesis. Thus, a glucose-induced uptake of Na1

through the mitochondrial Na1/Ca21 exchanger promotesCa21 extrusion and reverses the divalent cation uptake bythe mitochondrial Ca21 uniporter (Nita et al., 2014).After four decades of study on VGSC in pancreatic beta cells,

we have advanced in the understanding of how Na1 currentscontribute to cell depolarization, voltage and Ca21 oscillationsin beta cells, and, finally, in the promotion of glucose-inducedinsulin secretion (Fig. 2C). However, the regulatory pathwaysinvolving VGSC, from endocrine signals to epigenetic mech-anisms, are still an incipient field of research. The evidencesuggesting that Nav channels interact with metabolism in abidirectional way is fascinating, because the role of thesechannels is not yet entirely understood in metabolic diseasessuch as diabetes or the metabolic syndrome. Indeed, VGSCregulates the physiology of pancreatic beta cells throughmechanisms other than just a depolarizing effect, a reason toconsider them as potential drug targets for treating relatedpathologies.

Voltage-Dependent Calcium ChannelsVoltage-dependent calcium channels (VDCCs) mediate

calcium influx to the beta cells, coupling electrical signalingto Ca21-dependent protein-protein interactions, and enzy-matic responses, playing a crucial role in insulin secretion.Furthermore, Cav channels could also play roles in beta-celldevelopment, maturation, survival, growth, and death(Navarro-Tableros et al., 2007a; Hiriart and Aguilar-Bryan,2008; Yang et al., 2014a; Zhou et al., 2015).

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Molecular Structure of Cav Channels

VDCC consists of heteromeric complexes composed ofCava1, Cava2, Cavb-, Cavd-, and Cavg-subunits (Fig. 3).The Cava1 subunit forms the ion-conducting pore, and Cava2,Cavb-, Cava2d-, and Cavg-subunits regulate its conductivityand trafficking (Yang et al., 2014a). The Cava1 subunit isformed by four homologous domains that consist of six trans-membrane segments (S1 to S6), a membrane-associated poreloop (P-loop) between each S5 and S6 segments, three in-tracellular linkers (LI-II, LII-III, LIII-IV), and N and Ctermini. As mentioned above, S5 and S6 segments and theP-loop form the Ca21 conducting pore. The S4 segments workas the primary voltage sensor involved in the inactivation andthe reactivation of the channel (Catterall, 2000b). The three-dimensional structure of Cav channels has been described,using electron microscopy and X-ray crystallography. TheCavb subunit is intracellular; the Cavg subunit is formed byfour transmembrane segments, and the Cava2d is extracellu-lar and attached to the membrane through a disulfide linkageto the transmembrane delta subunit (Catterall, 2000b).We can classify VDCC according to different criteria. A

functional characterization considers their open probabil-ity, in low-voltage activated (T-type channels) and highvoltage-activated (L-, N-, P/Q-, and R-type channels). Asecond structure-based classification considers the type ofa1 subunits being expressed and subdivides Cav channelsinto three families, namely Cav1, Cav2, and Cav3. They areassociated with L-, P/Q-, N-, R-, and T- type Cav currents

(Hiriart and Aguilar-Bryan, 2008; Yang et al., 2014a; Zamponiet al., 2015). There are also several subtypes of L channels,depending on their alpha subunit, alpha1C (CACNA1C, Cav1.2)and alpha 1D (CACNA1D, Cav1.3). L-type Cav1.2 and Cav1.3channels are expressed in rats and mice and in rats, mice, andhumans, respectively. However, Cav1.3 contributes the most toglucose-stimulated insulin secretion in rats and humans andCav1.2 in mouse beta cells (Hiriart and Aguilar-Bryan, 2008;Rorsman et al., 2012).Beta cells from diverse species and cell lines express

different combinations of VDCC. For example, human beta-cell expression of CACNA1D mRNA exceeds CACNA1CmRNA by 60 times (Reinbothe et al., 2013). Although thesame predominance of Cav1.3 is observed in the rat, in themouse, Cav1.2 channels are dominant (Hiriart and Aguilar-Bryan, 2008; Rorsman et al., 2012). Cav3.1 and Cav3.2(T-type) currents are present in INS-1 cells, dog, human,and rat, but not in mouse. Moreover, human beta cells lackCav2.3 (N-type) currents (Taylor et al., 2005; Hiriart andAguilar-Bryan, 2008), and N-type Cav2.2 channels have beenidentified in adult rat beta cells (Navarro-Tableros et al.,2007b). Also, human beta cells express P/Q-type Cav2.1, andmouse beta cells show P/Q-type Cav2.1 and R-type Cav2.3channels (Yang et al., 2014a).To date, 10 mammalian a1 genes and four distinct Cavb

genes (Cavb1, Cavb2, Cavb3, and Cavb4) have been identifiedin beta cells. Rat and mouse native beta cells express b2 andb3 subunits, although b2 is more abundant than b3 in the rat

Fig. 3. Representation of the calcium channel subunit structure, signaling that regulates them, and hormone and drug effects on calcium influx throughcalcium channels. Greek letters depict each calcium channel subunit, and Roman numbers represent each domain in the alpha-1 subunit. PKA, proteinkinase A; PKB, protein kinase B; PKC, protein kinase C; PKG, protein kinase G; SNARE, solubleN-ethylmaleimide sensitive factor attachment proteinreceptor; Gbg, G protein subunits beta-gamma; CAMKII, calcium/calmodulin-dependent kinase II; GLP-1, glucagon-like peptide-1; NGF, nerve growthfactor; IL-1, interleukin 1; TNF-a, tumor necrosis factor alpha; IFNg, interferon gamma. Green arrows/names represent an increasing effect and redlines/names depict an inhibitory effect on calcium influx through Cav channels. Question mark symbols represent an unknown effect on calciumchannels. Magenta arrow describes physical interaction.

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(Yang et al., 2014a). To date, it has been suggested thatCavb2 and Cavb3 subunits modulate GSIS possibly by regu-lating the interactions between VGCCs and PKC isozymes(Rajagopal et al., 2014).

Pharmacology of Cav Channels in Pancreatic Beta Cells

Many pharmacological agents modulate the activity ofCav channels. For example, L-type Cav1 channels aresensitive to La31, dihydropyridines, alkyl phenyl amines,and benzothiazepines. Recently, it was observed thatthe natural polyphenolic flavonoid quercetin (3,39,49,5,7-pentahydroxyflavone) enhances L-type Cav1 current, alsostimulating insulin secretion in INS-1 cells. Quercetin inter-acts with L-type Cav1 in a different site from that of theagonist Bay K 8644 (1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methylester). The mechanism acts by shifting the voltage-dependentactivation toward negative potentials (Bardy et al., 2013).L-type Cav1 channels are the major contributors to the wholecell Cav current and insulin granule exocytosis in human, rat,and mouse beta cells. Despite Cav1.2 being the principalL-type Ca21 channel in mouse, Cav1.3 is the most importantL-type channel in rat and human beta cells. Cav1.3 plays arole in cAMP homeostasis through adenylate cyclase 1 andmay participate in insulin secretion in an indirect manner(Kitaguchi et al., 2013). Recent work suggests that Cav1.2 LII-III loop interactions may play a significant role in the glucose-stimulated excitability. Theymay regulate channel density onthe plasma membrane, the Ca21-induced-Ca21 release fromthe endoplasmic reticulum (ER), as well as the activity ofother channels such as Ca21-activated potassium channels(Catterall, 2000b; Wang et al., 2014).P/Q-type Cav2.1 channels are sensitive to v-agatoxin IVA,

N-type Cav2.2 channels are blocked by v-conotoxin GVIAbut are insensitive to dihydropyridines, alkyl phenyl amines,and benzodiazepines. R-type Cav2.3 channels are resistantto almost all the Cav channel blockers, but it is blocked by thetoxin SNX-482. Although SNX-482 has shown a high affinityfor Cav2.3 channels, it is capable of blocking other Cavchannels Yang and Berggren, 2006; (Catterall, 2011; Yanget al., 2014a).Calcium entry into the cell through P/Q-type Cav2.1

channels is the second most important entry route in rodentbeta cells after glucose stimulation. Nevertheless, P/Q chan-nels contribute in a similar fashion as L-type channels to thewhole cell Cav current in human beta cells (Yang andBerggren, 2006; Yang et al., 2014a). N-type Cav2.2 channelsare expressed in adult rat beta cells, although 50% of theprotein is distributed in the membrane and 50% at thecytoplasm (Navarro-Tableros et al., 2007b).T-type Cav3.1 and Cav3.2 channels play a significant role

in the regulation of insulin secretion. In rat beta cells, theyactivate around 240 mV and generate a hump at thebeginning of the whole cell I(Ca)-V relationship, with rapidinactivation and slow deactivation, and they may play the roleof a pacemaker at glucose-stimulating levels (Hiriart andMatteson, 1988; Hiriart and Aguilar-Bryan, 2008). T-type Cavcurrents have been suggested to be involved in the beta-cellresponse to cytokines (Wang et al., 1999). Finally, R-typeCav2.3 channels are expressed rodent but not human betacells (Hiriart and Aguilar-Bryan, 2008). They may play a role

in the second phase of insulin secretion by supplying newsecretory granules to the release sites (Jing et al., 2005; Yangand Berggren, 2006; Rorsman et al., 2012).

Endogenous Regulation of Cav Channels in the Beta Cell

Several mechanisms and pathways, including protein phos-phorylation, Ca21/calmodulin signaling, G protein-coupledreceptors, and phosphorylated inositol may endogenouslyregulate Cav channels activity and density in the beta-cellmembrane (Yang and Berggren, 2006). These regulatorymechanisms interact simultaneously to control insulin secre-tion in the body (Fig. 3).Serine/Threonine Protein Kinases, Phosphatases,

and Tyrosine Kinase Receptors. Pancreatic beta cellsand insulin secretion are regulated by cAMP and PKA. Cavchannels possess several protein phosphorylation sites. Dif-ferent studies have shown the regulation of Cav channels byPKA. An increased Ca21 influx through VDCC is observedwith cAMP analogs or activators of PKA, and this effectdisappears with channel blockers (Henquin and Meissner,1983, 1984). Moreover, mouse beta cells showed not only anincreased exocytotic capability assessed by capacitance mea-surements after PKA activation (Gillis and Misler, 1993) butan increased amplitude in L-type Cav currents in a dose-dependent manner after cAMP-analog exposure (Kanno et al.,1998; Yang and Berggren, 2006). More recently, it has becomeclear that PKB may also participate in GSIS. PKB mayupregulate L-type calcium channel activity via phosphoryla-tion of the Cavb2 subunit, which promotes calcium channeltrafficking to the membrane (Cui et al., 2012).Effects on Cav by PKC activation are controversial. PKC

depletion fromRINm5F cells downregulates L-, P/Q-, and non-N-type Cav currents (Yang et al., 2014a), and acute activationof PKC produces an increase in Cav1 channels. Thus, PKCmodulation has been suggested to play a tonic role inmaintaining Cav channel function (Yang and Berggren,2006). Pancreatic beta cells express all PKG isoforms (PKG1a,PKG1b, and PKGII) and all four Ca21/calmodulin-dependentprotein kinase II (CaMKII) isoforms (Easom, 1999). Further-more, activation of PKG in rat beta cells by cGMP increasedCa21 influx through Cav channels. Nevertheless, furtherinvestigation is needed, because cGMP can produce a directeffect on Cav channels (Yang and Berggren, 2006; Yang et al.,2014a).Also, regulation of Cav channels by calcium/calmodulin-

dependent kinase II (CaMKII) is not clear. Although non-specific CaMKII inhibitors, namely KN-62 and KN-93, couldinhibit Cav channels, it has been suggested that both may actdirectly on beta-cell Cav channels (Yang and Berggren, 2006).Recently, a reduction in insulin secretion and Ca21 influxthrough Cav channels was observed in islet beta cells afterCaMKII inhibition (Dadi et al., 2014).Several types of protein phosphatases are present in beta

cells. Experiments using okadaic acid to inhibit proteinphosphatases in mouse and rat beta cells have shown a smallincrease in basal conditions but a significant increase in Cavchannel activity with a simultaneous activation of PKA. Thus,it was suggested that Cav channels from mouse beta cellsshow low phosphorylation level in basal conditions because ofa dominant effect of tonically activated phosphatases that

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surround Cav channels over the protein kinases activity(Ammälä et al., 1994).Former work in our laboratory showed that TrkA tyrosine

kinase receptors regulate rat beta-cell Cav channels. Acuteincubation of rat beta cells with nerve growth factor (NGF)induces activation of Cav1 channels probably by tyrosinephosphorylation that produces increased Ba21 currents(Rosenbaum et al., 2001). Moreover, long-term exposure toNGF potently increases Ba21 currents due to an increasedCav1 synthesis (Rosenbaum et al., 2002). Insulin receptorsignaling might also upregulate Cav channel activity. Indeed,acute incubation of mouse beta cells with an insulin mimeticdrug L-783,281 increased intracellular Ca21 concentration ina dose-dependent manner, and this effect was partiallyblocked by nifedipine (Roper et al., 2002).G Protein-Coupled Receptors. The Cav2.1 and Cav2.2

channels can be modulated by direct membrane-interactionwith G proteins. The Gbg subunits play a fundamental role inthe regulation of Cav channels. The Gbg subunits bind to theintracellular linkers, LI-II of Cav2.1 and Cav2.2 channels, andto the LI-II of Cav1.3 channels. This interaction is character-ized by a positive shift in the voltage dependence and aslowing of channel activation. Furthermore, the N and Ctermini of the Cava1 subunit are also involved in Gbg binding(Dolphin, 2003; Yang et al., 2014a). Moreover, it was observedthat Cav1.2 channels are negatively regulated by a directmembrane-interaction with G proteins (Ammälä et al., 1992;Ivanina et al., 2000).It is well accepted that chronically increased concentrations

of free fatty acids impair beta-cell function and reduce insulinsecretion (Zraika et al., 2002). However, some observationssuggest that prolonged exposure to high levels of someunsaturated free fatty acids could protect beta-cell functionand increase GSIS (Tuo et al., 2012). It was suggested that themedium- and long-chain free fatty acid receptor GPCR40couldmediate this effect by activation of theGaq protein, Ca21

release from the endoplasmic reticulum, and influx via L-typeCa21 channels (Fujiwara et al., 2005).Glucagon and Glucagon-like Peptide Receptors. It

has also been speculated that activation of glucagon receptorsregulate Cav channels. Experimental evidence for a regula-tory role of the G-coupled glucagon receptor over Cav channelscomes from experiments in the insulin-secreting HIT cells.These cells showed an increase in intracellular Ca21 concen-tration in response to glucagon that was blocked by eitherchelation of extracellular Ca21 or application of Cav1 channelblockers (Prentki et al., 1987; Yang et al., 2014a).Glucagon-like peptides (GLP-1) potentiate nutrient-

induced beta-cell insulin secretion and proliferation anddecrease their apoptosis (Phillips and Prins, 2011). The GLP-1effect on mouse beta-cell insulin secretion is in part due to aslower time-dependent inactivation of Cav currents. Similareffects have been observed on Cav1 channels in the rat andhuman pancreatic beta cells (Yang et al., 2014a). Other workshave suggested that PKA activation may modulate the VDCCspore activity. Moreover, it was observed that PKA inhibitorsblock the GLP-1-mediated increase of Ca21 currents (Meloniet al., 2013). Also, Melissa and collaborators suggested thatthe intracellular II-III loop domain of Cav1.2, and 1.3 maytarget these channels to specific membrane microdomains,allowing their interaction with proteins involved in GLP-1signaling (Jacobo et al., 2009).

Somatostatin Receptors. Activation of somatostatin re-ceptors decreases Cav channel activity via a pertussis toxin-sensitive G protein in HIT and RINm5F cells (Prentki et al.,1987). Recent work determined that somatostatin reducesGSIS in mouse islets partially by modulating L-type Ca21

channels and suggested that somatostatin may exert thiseffect through a Gbg protein-dependent mechanism (Schwetzet al., 2013).Catecholamine and Acetylcholine Signaling. Para-

sympathetic and sympathetic innervation of islets modulatesinsulin secretion. Sympathetic innervation activates adreno-ceptors alpha2 and decreases insulin secretion and para-sympathetic innervation releases acetylcholine, whichactivates muscarinic receptors and stimulates insulin secre-tion (Hiriart et al., 2014). Stimulation of mouse beta-cellalpha2 adrenoceptors using clonidine inhibits Cav channelactivity and insulin secretion. Moreover, this effect is abol-ished by the selective alpha2 antagonist yohimbine. However,further work is needed to describe the mechanism by whichalpha2 adrenoceptors modulate beta-cell Cav channels (Yangand Berggren, 2006). The sympathetic nerve terminals alsorelease galanin. This neuropeptide also inhibits Ca21 currentsand thus insulin secretion in mouse beta cells; this effect couldbe mediated by a Go2-alpha protein (Tang et al., 2012).Interestingly, pancreatic beta cells synthesize and store

dopamine within their secretory granules. Also dopamine iscosecreted with insulin and exerts an autocrine or paracrinenegative feedback loop on beta-cell insulin secretion, actingthrough D2 and D3 receptors. Furthermore, D3 signalingactivates Gbg proteins, which may negatively regulate both,directly and indirectly, Cav1.2 channels activity (Ivaninaet al., 2000; Ustione et al., 2013).Little information exists on acetylcholine signaling in beta

cells. Despite the fact that the parasympathetic neurotrans-mitter acetylcholine stimulates insulin secretion, it alsoinhibits the Cav channel activity in mouse beta cells (Gilonet al., 1997). However, studies in HIT cells have observed thatmuscarinic receptors increase the amplitude of whole cell Cavcurrents and the Cav1 channel open probability (Yang andBerggren, 2006).Exocytotic Proteins. Exocytotic proteins such as syn-

taxin 1A, SNAP-25, and synaptotagmin also interact withL-type VDCC. Beta cells predominantly express Syn-1A,Syn-2, and Syn-4 in the membrane and Syn-3 in the secretorygranules. This interaction tranduces bidirectional signalingthat modulates Cav1 channel activity and the exocytoticmachinery and could mediate exocytosis of predockedinsulin-containing vesicles during the first phase of GSIS(Yang et al., 2014a). Syn-1A deletion in mice blunts the firstphase of GSIS (Kang et al., 2002; Xie et al., 2016). Further-more, syntaxin 3A binds and modulates Cav2.3 channelopening, as well as the fusion of newcomer insulin vesiclesduring the second phase of GSIS. Syn-3 decreases with theRNAi in INS-1 cells after the second phase of insulin secretion.Moreover, overexpression of Syn-3 also inhibits L-type chan-nels. Furthermore, Syn-3 depletion in INS-1 832/13 cellsincreases Cav activity. Syn-3 preferably binds to R-typecalcium channels and a lesser extent to L-type Ca channels,which show a preference for Syn-1A (Xie et al., 2016).Cytokines. Cytokine action in beta cells could be linked to

calcium influx through VDCC. Some studies observed thatblocking L-type calcium channels prevented IL-1B-induced

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apoptosis in rodent and human islets, although ERK1/2, p38,and JNK pathways may also be involved (Maedler et al., 2004;Fei et al., 2008; Pratt et al., 2016). Additional data fromnonobese diabetic mice showed a beta-cell increased expres-sion of the T-type calcium channel, which results in elevatedbasal intracellular Ca21. In addition, the treatment of mousebeta cells with a cytokine cocktail induced expression ofthe T-type calcium channel (Wang et al., 1996). Moreover,mouse islet exposure to a low-dose cytokine combination,namely, tumor necrosis factor-alpha, interleukin-1 beta, andinterferon-gamma, induced beta-cell dysfunction, partly dueto an increased calcium influx through L-type calcium chan-nels (Dula et al., 2010). However, cytokine-induced changes inVDCC activity and insulin secretion were not apparent. Earlystudies observed that interleukin-1 could inhibit glucose-stimulated calcium influx in rat islets (Wolf et al., 1989), butother studies showed the opposite effect (Ramadan et al.,2011). This effect is probably mediated by diacylglycerolproduction and protein kinase C activation (Welsh et al.,1989; Eizirik et al., 1995). The variability observed in thesestudies may reflect dosage dependence and the differentcocktail combinations used in experiments (Ramadan et al.,2011).

The Role of Cav Channels in the Pathophysiology of MS andT2DM

VDCC alterations, because of mutation, the downregulationof their expression, activity, and/or density at the membrane,could lead to beta cell dysfunction (Yang and Berggren, 2006;Velasco et al., 2012). Changes in calcium influx throughVDCCplay a vital role in beta-cell dysfunction inmetabolic syndrome(MS) and diabetes models. In an MS rat model induced byconsumption of a 20% sucrose solution during 24 weeks,changes in whole cell calcium influx were observed, whichmay be related not only to beta-cell hypersecretion butdysfunction and exhaustion (Velasco et al., 2012). Additionalstudies have shown that a 48-hour islet exposure to 28 mMglucose increased insulin secretion due to an increasednifedipine-sensitive VDCC influx (Qureshi et al., 2015).It is well accepted that beta-cell chronic exposure to high

long-chain free fatty acids contributes to increasing basalinsulin secretion and, at the same time, reduces GSIS duringobesity (Zraika et al., 2002). In part, these effects involve areduced calcium influx through VDCC, associated with anincreased activity of the membrane metalloendopeptidaseneprilysin (Zraika et al., 2013). Other groups have found thatbeta-cell long-term exposure to free fatty acids results inselective suppression of exocytosis elicited by brief depolar-izations. They suggested that depolarization-induced Ca21

influx is limited to discrete areas within beta cells due tocalcium channel aggregation and that FFAs induce a diffusedCa21 influx along the membrane surface. This effect couldimpair insulin exocytosis, because release-competent gran-ules may not be exposed to exocytotic local levels of Ca21

(Rorsman et al., 2012). Furthermore, two single-nucleotidepolymorphisms in the CACNA1D gene (Cav1.3) reduce itsmRNA expression, impair insulin secretion, and are associ-ated with type 2 diabetes (Reinbothe et al., 2013). Finally,blocking L-type calcium channels with diazoxide could inhibithyperglycemia-induced beta-cell apoptosis (Zhou et al., 2015).

ConclusionsElectric activity in beta cells is essential for insulin secre-

tion. A great deal is known about the different channels thatparticipate in depolarization and action potentials. However,there are other ion channels in the plasma membrane of betacells and in the organelles that are not so well understood,such as chloride and potassium channels, which appear to beinvolved with membrane repolarization. Studies of channelo-pathies for KATP have provided important insight and arebeginning to emerge for other channels. In the future, it will beimportant to develop specific drugs for different channels thatcould be used to directly test functional roles in beta cells andinsulin secretion. In addition, it will be important to continue toinvestigate the epigenetic changes of the channels and exploretherapeutic strategies to reverse associated deficits.

Acknowledgments

ASPET thanks Dr. Katie Strong for copyediting of this article.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Velasco,Díaz-García, Larqué, and Hiriart.

Performed figures: Velasco, Díaz-García, and Larqué.

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