Potassium Channels

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Missy Cavallin September 14, 2007. Potassium Channels. Outline. Types of K + channels Voltage-gated Functional roles Nomenclature Structure Activation Inactivation Assigned Experimental Papers (structure, voltage sensor, inactivation) ‏. Types of K + Channels. Inward Rectifying - PowerPoint PPT Presentation

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  • Missy CavallinSeptember 14, 2007Potassium Channels

    Doyle et al. (1998) Science 280:69-77

  • OutlineTypes of K+ channelsVoltage-gatedFunctional rolesNomenclatureStructureActivationInactivationAssigned Experimental Papers (structure, voltage sensor, inactivation)

  • Types of K+ ChannelsInward RectifyingCa+2 sensitiveATP-sensitiveNa+ activatedCell volume sensitiveType AReceptor-coupledVoltage-gated

    Armstrong and Hille (1998) Neuron 20:371-380

  • Inward Rectifying (KIR)2 transmembrane regions (M1 and M2)4 subunits form poreP region between M1 and M2Regulated by concentration of extracellular potassiumActivity dependent on interactions with phosphatidylinositol 4,5-bisphosphate (PIP2)Blocked by external Ba+

  • Functions of KIRMaintain membrane resting potential near EKnon-conducting at positive membrane potentialsContributes to cell excitability

  • Ca+2 sensitive4 protein subunitsSelectivity filter on external surfaceRCK domains act as gate2 Ca+2 ions bind to RCK to regulate gate3 types of channelsHigh conductance (BK): 100-220 pSIntermediate conductance (IK): 20-85 pSSmall conductance (SK): 2-20 pS

  • Functions of Ca+2 sensitiveGenerate membrane potential oscillationsAfterhyperpolarization

  • ATP-sensitive2 Transmembrane regions4 subunitsInhibited by ATPInwardly rectifyingNot voltage dependent

  • Functions of ATP-sensitiveCouples K+ conductance to cell metabolic stateResponds to metabolic changes: e.g. senses glucose concentration in -cellsSenses intracellular nucleotide concentrations

  • Na+ activatedVoltage-insensitiveBlocked by Mg+2 and Ba+2

  • Cell volume sensitiveActivated by increase in cell volumeBlocked by quinidine, lidocaine, cetiedil

  • Type ATetramer of -subunits and intracellular -subunitsRapid activation and inactivationInactivation may involve -subunitsPossible role in delaying spikes by regulation of fast phase of action potentials

  • Receptor-coupledMuscarinic-inactivatedSlow activationNon-inactivatingNon-rectifyingAtrial muscarinic-activatedInward rectifying

  • Voltage-gated channels: FunctionRegulate resting membrane potential toward EKControl shape and frequency of action potentialsKeep fast action potentials shortTerminate intense periods of activityTime interspike intervalsLower the effectiveness of excitatory inputs on a cell when openDelayed rectifier type expressed in axonsDelayed activation and slow inactivation (shape action potential)

  • Nomenclature of voltage-gated K+ channels

    Gutman et al. (2005) Pharmacol Rev 57:473-508

  • Voltage-gated: Structure6 transmembrane (TM) regions (S1-S6)Principal subunitsHomo- or heterotetramersS5 & S6 of each subunit surround poreAuxiliary subunitsRegulate channel activity

    Gulbis et al (2000) Science 289:123-127

  • More StructureS4 segment has multiple positively charged amino acids (arginine or lysine)voltage sensorP loop between S5 & S6 (selectivity filter)G-Y-G sequence required for K+ selectivityT1 domain: tetramerization domain; connects b subunits to a subunits

  • K+ movement through channelK+ attracted to negative charge of helices near selectivity filterK+ becomes hydrated before exiting channel

    Doyle et al. (1998) Science 280:69-77

  • Voltage-gated: ActivationActivated by depolarizationVoltage sensor (S4)Translocation of charges on S4Helical screwLateral movement of crossed helicesRocking motion at interface of 2 domains

  • Voltage-gated: InactivationN-type: ball-and-chainAmino acids at N-terminus occlude intracellular channel poreRapid inactivationC-typeConformational changes at selectivity filter or extracellular entrance to channelSlow inactivation

    Kurata and Fedida (2006) Progress in Biophysics and Molecular Biology 92:185-208

  • Rasmusson et al (1998) Circ. Res. 82:739-750

  • Paper 1Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K+ Channel

    Stephen B. Long, Ernest B. Campbell, and Roderick MacKinnon

    Science (2005) 309:897-903

  • MethodsKv1.2 with b2 subunit from rat brain expressed in yeastX-ray crystallography2.9 resolution

  • Figure 1 A) Electron density map = blue mesh; final model trace = yellow B) Crystal lattice structure of channel: transmembrane segments of a subunit = red; b subunit + T1 = blue; unit cell = black box

  • Kv1.2 structural features4-fold symmetry (tetrad axis of unit cell)Dimensions of tetramer: ~135 x 95 x 95 Length of transmembrane segments: ~ 30 (approximately thickness of lipid bilayer)

  • Figure 2 A) Side view of ribbon model of channel with 4 subunits of unique color. NADP+ cofactor = black sticks B) 1 subunit from panel A illustrating S1-S6 as well as N- and C-termini C) Looking into pore from extracellular side to show interactions of 4 subunits.

  • Figure 3 A) Side view of 2 subunits of three voltage-gated K channels: Kv1.2 = red; KcsA = gray; KvAP = blue; outer = S5 of Kv1.2; inner = S6 of Kv1.2 B) Top view into poreNote the overlap of the structures (conserved).

  • Paper 2How Does a Voltage Sensor Interact with a Lipid Bilayer? Simulations of a Potassium Channel Domain

    Zara A. Sands and Mark S.P. Sansom

    Structure (2007) 15:235-244

  • How is a positively charged S4 helix able to stably span the lipid bilayer?Used molecular dynamics simulations to test the interactions of the voltage sensor domain of KvAP (archaebacterial) with an artificial phosphatidylcholine (PC) bilayer or with a more natural PC + phosphatidylglycerol (PG) bilayerComputer modelingExtended time (50 ns) vs. other models (20 ns) to increase sampling

  • ResultsPenetration of water into center of voltage sensor and bilayerLocal deformation of bilayer due to interactions of lipid head groups with arginine side chains of S4 subunitElectrostatic field is focused at center of bilayerNumerous hydrogen bonds between phosphate head groups of lipids with arginine residues of S4 subunit

  • Figure 1 Lipids interact strongly with and are drawn into voltage sensor causing changes in PC bilayer conformation. A) Voltage sensor shown as ribbon (S1-3 = gray; S4 = magenta); lipid phosphate head groups colored based on z coordinate (red = extracellular; blue = intracellular). Arrow shows phosphate that is pulled away from surface. B) Arginines on S4 interactions with lipid phosphate groups.

  • Figure 2 Shows lipid bilayer compression when voltage sensor present vs. respected control bilayers without proteins. (distance between upper and lower phosphate atoms)

  • Figure 3 There are more hydrogen bonds formed in PC bilayer. Although both bilayers have hydrogen bonds between S4 arginines and lipid phosphate groups or water.

  • Figure 4 A and C) red = water; blue = lipid B) blue = cationic side chain; red = anionic side chain

    PC/PG overall has more hydrogen bonds.

    Increased hydrogen bonds at termini may stabilize charged S4 in bilayer.

    Lack of water to hydrogen bonds at R133 indicates prevention of water penetration.

  • Figure 6 A) Water in cavities of of voltage sensor but not at constriction point. B) Model of average pore radius. C) Electrostatic potential distribution ( 120 mV). Note the focus of electrical potential around salt bridge at constriction point.

  • ConclusionsHydrogen bonding may help to stabilize voltage sensor in membraneCompression of bilayer can decrease the distance necessary for the movement of C-type inactivation of potassium channels

  • Paper 3Slow Inactivation in Voltage Gated Potassium Channels is Insensitive to the Binding of Pore Occluding Peptide Toxins

    Carolina Oliva, Vivian Gonzalez, and David Naranjo

    Biophysics Journal (2005) 89: 1009-1019

  • Toxin binding to K channels and Slow (C-type) InactivationSensitive to K+ or tetraethylammonium (TEA) in poreMutations of external vestibule alter both processesDoes toxin binding interfere with C-type inactivation?

  • Figure 1 B) Amino acids important for toxin binding (left) and slow inactivation (right) overlap. red = scorpion toxin; yellow = conotoxin; orange = both; blue = slow inactivation

  • MethodsOocytesWhole cell recordingOutside-out patch recordingToxinsConotoxin (k-PVIIA)Charybdotoxin (CTX)

  • ResultsRate of inactivation and recovery are toxin insensitive Inactivation does not alter toxin binding site

  • Figure 2 Whole cell voltage clamp. A) Conotoxin (thick trace) decrease current amplitude and causes slight delay in activation. B) Time course of ratio of currents in panel A. Toxin binding (and current ratio) reached steady state within recording interval.

  • Figure 3 and Table 1 show that the inactivation kinetics are not affected by conotoxin in spite of change in current amplitude. Therefore, conotoxin binding is independent of slow inactivation. Similar results were shown for CTX (Fig. 6). There are differences between whole cell vs. outside out patches, but not with regards to toxin effects.

  • Figure 5 A) Conotoxin decreases current amplitude in response to voltage steps. B) IV and GV relationships. Voltage shift does not change in the presence of toxin (bottom). DV = -18 mV for no toxin; DV = -21 mV with toxin

  • ConclusionsInactivation kinetics were not affected by toxin bindingBound toxin does not hinder conformation change involved with slow inactivation