Neurobiology Block 5

7/29/2019 Neurobiology Block 5

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Lecture 5

Ion channels and Resting

Potential


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Equilibrium potential at room temp for K+ in the

giant axon of the squid:

E = RT/zF ln ([K+]o/[K+]i) = 25.7mV ln(20/400) =

appr. -75 mV

R = gas constant = 8.314 J mole -1 K-1

T= 298 K

z = 1 as for [K+]F = Faraday constant = 9.65 x 10000C/mole = charge carried per

mole of electrons

-> 2.3 RT/zF = 58 mV

[K+] inside squid axon = 400 mM

[K+

] outside, 20 mM


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A 0 mV large, outward

B - reduced, outward

C more - further reduced

D equilibrium none

potential inward I = outward I

K

intracellular extracellular

K

K K

K K

- +

--

+

+

K K

- +

- +

Time membrane netpotential current

The equilibrium potential of an ion is determined by the

ions concentration gradient

At Equilibrium Potential:

Chem. driving force+ elect. driving force = 0

(they are equal but in opposite direction)

+-

chemical driving

force

electrical driving

force

K+ current


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Squid axon K+ Na+ Cl-

Cytoplasm (mM) 400 50 52Extracellular (mM) 20 440 560

Squid axon at rest: PK:PNa:PCl =1.0 : 0.04 : 0.45

Can you calculate the resting membrane potential?

What would the resting membrane potential be if the resting channels were

only permeable to Na ions?

Example: calculate the membrane potential of the squid axon

Emem = RT/F * lnPNa+[Na+out] + PK+[K+out] + PCl-[Cl-in]

PNa+[Na+

in] + PK+[K+

in] + PCl-[Cl-out]


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Recording the membrane potential

Resting membrane potential = electrical potential across membrane atrest: Vm = Vin Vout

Glass microelectrodes are filled with salt solution and connected via amplifier to

oscilloscope.

Extracellular electrode serves as reference

potential outside of the cell = 0 mVOne electrode is inserted into the cell to measure the membrane potential on inside of the

cell


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glial cells nerve cellsChannels: forK+ only forK+, Na+ and Cl-

A membranes overall selectivity for individual ion is determined by the relativeproportions of various types of ion channels in the cell that are open

extracellular

cytoplasmic

K+ Na+ Cl- K+ Na+ Cl-

How do ionic gradients contribute to the resting

membrane potential?


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Determined by:1. Ionic selectivity of resting channels

2. Concentration gradients of permeant ions

3. Na/K ATPase

Players that contribute to the resting potential:

(1) Two-pore domain leak K+ channels, some voltage-gated K+ channels

(2) Hyperpolarization-activated and cyclic-nucleotide-activated channels (HCN

channels, corresponding current is typically termed Ih)

(3) Inwardly rectifying K+ (IRK) channels (inward > outward current)

(4) Persistent sodium current (~1-3% of sodium channels do not inactivate at

any given time)

(5) Extrasynaptic GABAA receptors (to be discussed later)

(6) Electrogenic Na+/K+ pump

Resting membrane potentials


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Passive influx of Na and efflux of K is balanced by active pumping of the ions.

Na-K pump moves Na and K against their net electrochemical gradients:(1) Extrudes 3 Na ions from cell and takes in 2 K ions /each cycle (-> net outward current

that contributes to negative resting membrane potential)

(2) Requires energy, hydrolysis of ATP.

Na+ - K+ pump prevents the ionic

gradients from dissipating by diffusion

of ions across the membrane through

the resting channels, and thus

maintains the resting membrane

potential.

At the resting potential, the ion gradient

across the membrane has the tendency

to be reduced by leaky channels but

is constantly regenerated by the Na+ -

K+ pump. The cell is in a steady state.

How are ionic gradients maintained ?


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Channels vs. Transporters (pumps)

Channel

Passive ion conduction

Large conductance

Uses ion gradients

Used for fast signaling

Hundreds of Genes

Transporter

Active ion transport

requires energy

Small conductance Establishes ion gradients

Background process that

maintains environment for

fast signaling Hundreds of genes


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General properties of ion channel gating

Gating of ion channels requires energetic input

Major gating mechanisms: Voltage (energy provided by changes in transmembrane potential)

Used to regulate intrinsic excitability and generate action potentials

Ligands (energy provided by binding of a ligand)

Neurotransmitter gated ion channels at synapses

Link channel opening to the presence of intracellular signals

(G-proteins, calcium, cyclic nucleotides etc.)

Sensory (energy provided by sensory stimuli)

Mechanosensitive channels

Temperature sensitive channels

Acid sensing channels

-70 mV 0 mV


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KIR

K2P

(X2)

ORAIGLU

VSD

P

KV

KCa

TRPCATSPER

HCN

CNG

TPC (X2) (X4)

NaV

CaVNALCN

P2X

ASIC C-Loop ReceptorsCLC

A B

C D E

Cartoon Representations of Ion Channel Structures

Neurotransmitter-gated Voltage-gated


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Channel Pore Symmetry

Voltage-gated Ion Channels 4-fold, 1-4 subunits.

Voltage-gated K+ channels contain 4 homologous

subunits (homomeric or heteromeric)

Voltage-gated Na+ and Ca2+ channels contain one

subunit with 4 homologous motifs

Glutamate neurotransmitter receptors 4-fold, 4 subunits (usually heteromeric)

Probably evolved from K+ channels

C-loop neurotransmitter receptors GABA-A, Glycine, nicotinic Ach, 5-HT 3 (serotonin)

5-fold symmetry, 5 subunits, heteromeric


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Neurotransmitter-gated ion channels vs. G-

protein-mediated neutransmitter responses

Glutamate gated-ion channels(18 genes):

AMPA receptors

Kainate receptors

NMDA receptors

GABA-A receptors (19 genes)

nACh receptors (16 genes)

Glycine receptors (5 genes)

5HT3 receptor (3 genes)

Metabotropic glutamate receptors

GABA-B receptors

mACh receptors (muscarinic)

5HT receptors (not 5HT3)

Dopamine receptors

NE receptors

+ many others

100+ genes

Typically modulate the

activity of ligand- or

voltage-gated ion channels

NT-gated ion channels are used

for fast synaptic potentials

NT-gated GPCRS are usedfor slower signaling


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Represents the non-redundant channel

set required for animal physiologymost are expressed in the nervous

system

Consists of 46 separately conserved ionchannel types 34 are from the voltage-gated channel

superfamily (which includes someligand-gated channels)

14 K+ channel families

(145-380 genes/animal genome)

A Fundamental Channel set is shared by

all animals with nervous systems


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Evolutionary History of the

Voltage-gated Cation Channel

Superfamily

Out

In

Gating Domains


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Ion Channels and the Human Genome

~300 mammalian genes encode ion channel pore-

forming subunits (1% of the Genome)

Distributed throughout genome

Many channels also contain modulatory subunits that

effect gating and localization, but not conduction


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?

?

ProkaryotesSingle-Cell

Eukaryotes

Basal

MetazoansBilateria Vertebrates Mammals

CaV

NALCN

NaV

TPC

TRP

CATSPER

Classic KV

KV7

KCa

2,3

KCa

1,4,5

CNG

HCN

KV10-12

KIR

K2P

GLUR

Most mammalian channel

genes were produced byDuplications that occurred

early in the vertebrate lineage

The mouse and human channel

sets are virtually identical


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Functional analysis of channels

Patch clamp recording technique

What do channel currents look like?


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Patch Clamp Recording Configurations

Voltage clamp control voltage

and measure current

Used to study channel gating

Current clamp control current

and measure voltage

Used to study neuronal activity

Glass micropipette

connected to amplifier


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Single channel analysis of nACh receptors

Fig. 6-3

Channels open in an all or none

fashion

Probability of opening or

closing is controlled by

gating stimuli


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1 channel

3 channels

dozens ofchannels

hundreds ofchannels

time (ms)

I (pA)

Patch clamp recording is highly scalable:

Patches containing single channels

Patches containing many channels (macroscopic currents)

Whole cell currents

Many types of channels in neurons

Single type by overexpression in vitro


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The I/V relationship of ion channels

Channels that exhibit a linear I/V relationship are resistor

like (= Ohmic) (usually ligand gated or sensory channels)

Many ion channels conduct the current more easily in one

direction than in the other -> Rectifying ion channels

Rectification can be caused by selectivity filter properties (preferential binding site

availability from one side of the membrane) or voltage gating (only open at some

voltages)


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Macroscopic voltage clamp recordings allow quick

measurement of voltage-gated channel kinetics and

open probability


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Ion Channels and Diseases

Disruption of ion channel signaling is a

significant cause of inherited neurological

disease.

Epilepsy can be caused by gain of function inexcitatory channels or loss of function in

inhibitory channels.

Migraine, ataxia and neuropathic pain are otherexamples of channelopathies.

Neurobiology Block 5

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