Lecture 7: Ion channels

What are ion channels and why are they ideal mediators for fast neuronal signaling? What types of ion channels are there?
Diversity in selectivity and gating mechanisms

How do ion channels work and how do we study their function?

Ion channels are integral membrane proteins that provide a gated pathways for movement of ions across cell membranes
ClClClClCl-

Out
Cl-

ClCl-

Cell membrane has hydrophobic core that is impermeable to ions

ClCl-

Cl-

In
Cl-

Movement of charged ions across the cell membrane is the basis of electrical signaling in the nervous system

Why does the nervous system use ion channels and electrical signaling?
Speed! Fast signals = fast behaviors
Current mediated by ion channels causes rapid change in membrane potential (up to 500 mV/msec)
It is the change in membrane potential that is the communicated neuronal signal, not the ion current itself.

In most cases conductance is limited by diffusion only -> much faster than most enzymatic reactions Most ion channels pass small inorganic ions present in high concentrations (Na+, K+, Ca2+ and Cl-).
These ions are highly mobile and present in concentrations that allow large, fast currents.

Note: ion channels are present in all living cells and are used for everything from electrical signaling and muscle contraction to fluid transport and maintaining osmotic balance.

General properties of ion channel conduction

Ion channels are selective for specific ions Flow of ions through channels is passive

The direction of ion flow is determined by electrochemical gradients for permeable ions
The gradient is determined by ion concentration inside and outside the cell, and the transmembrane electrical potential

Ions have equilibrium potentials (Eion) where these gradients are in balance and there is no net current flow.
An open ion channel will drive the membrane potential to the equilibrium potential of the ions that permeate it.

K+ and Cl- channels are used to hyperpolarize (silence) neurons Na+ and Ca2+ channels are used to depolarize (excite) neurons

Calculating Reversal Potential for an Ion

The Nernst Equation
Erev = RT/zF * ln [Ionout]/[Ionin] z = valence F = Faradays constant (96485 C/mol) R = Gas constant (8.314 J/K*mol) T = temperature (K) Erev is in volts, or J/C
RT/F = 25.7 mV at Room Temp (25C)

Calculating Reversal Potential of the Cell Membrane

GoldmanHodgkinKatz equation (Goldman Equation)
Emem = RT/F * ln PNa+[Na+out] + PK+[K+out] + PCl-[Cl-in] PNa+[Na+in] + PK+[K+in] + PCl-[Cl-out]

P = permeability F = Faradays constant (96485 C/mol) R = Gas constant (8.314 J/K*mol) T = temperature (K) Erev is in volts, or J/C

Selectivity Notes
Size of hydration shell, rather than of ion itself determines ion selectivity Among different ions with same charge, smaller ions exhibit a stronger electric field and therefore a stronger electrostatic attraction for water -> Na+ has larger shell of water than K+ The smaller the ion the smaller its mobility! Ion channel selectivity is often achieved through precise replacement of the hydration shell at a selectivity filter.

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

-70 mV
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.)

0 mV

Sensory (energy provided by sensory stimuli)

Mechanosensitive channels Temperature sensitive channels Acid sensing channels

Gating notes: Ion channels mediating light responses are actually ligand-gated ion channels
Vertebrate visual perception cGMP-gated cation channels respond to cGMP changes mediated by light-activated GPCRs (opsins) Vertebrate circadian system PLC-gated cation channels respond to activation of the Gq/PLC pathway by the circadian opsin melanopsin.
Wavelength dependence of TRPC3 currents activated by Melanopsin

Gating notes: desensitization and inactivation

In response to prolonged stimulation, many ion channels desensitize or inactivate
5 nA 40 ms

-> channel closes and goes into a functional state that resists stimulation (refractory state) -> duration of channel opening in many cases is brief even in the presence of a lasting stimulus

Gating notes: drugs and endogenous ligands can bind to channels with distinct pharmacological effects

Binding and action can be reversible or irreversible Agonist binds to same site as endog. ligand and activates (opens) ion channel Antagonists binds to same site and competes with agonist but has no activating function Allosteric modulators (B1) act at sites that differ from normal ligand binding site and allosterically change the affinity of the agonist and thereby modulate channel function

Figure 6-8

Cartoon Representations of Ion Channel Structures

A B
VSD
P

KIR

K2P (X2)

KV KCa TRP CATSPER HCN CNG

TPC (X2)

(X4) NaV CaV NALCN

GLU

ORAI

P2X ASIC

CLC C-Loop Receptors

Neurotransmitter-gated

Voltage-gated

Neurotransmitter-gated ion channels vs. Gprotein-mediated neutransmitter responses

NT-gated ion channels are used for fast synaptic potentials Glutamate gated-ion channels (18 genes): AMPA receptors NT-gated GPCRS are used for slower signaling Metabotropic glutamate receptors GABA-B receptors mACh receptors (muscarinic) 5HT receptors (not 5HT3) Dopamine receptors NE receptors + many others

Kainate receptors
NMDA receptors GABA-A receptors (19 genes)

nACh receptors (16 genes)

Glycine receptors (5 genes) 5HT3 receptor (3 genes)

100+ genes Typically modulate the activity of ligand- or voltage-gated ion channels

A Fundamental Channel set is shared by all animals with nervous systems

Represents the non-redundant channel set required for animal physiology most are expressed in the nervous system Consists of 46 separately conserved ion channel types
34 are from the voltage-gated channel superfamily (which includes some ligand-gated channels) 14 K+ channel families

(145-380 genes/animal genome)

Evolutionary History of the Voltage-gated Cation Channel Superfamily

Out
In
Gating Domains

Ion Channels and the Human Genome

~300 mammalian genes encode ion channel poreforming subunits (1% of the Genome)

Distributed throughout genome

Many channels also contain modulatory subunits that effect gating and localization, but not conduction

Prokaryotes

Single-Cell Eukaryotes

Basal Metazoans

Bilateria

Vertebrates

Mammals
NALCN CaV

NaV

Most mammalian channel genes were produced by Duplications that occurred early in the vertebrate lineage

TPC

TRP

CATSPER

Classic KV

KV7

KCa2,3 KCa1,4,5 CNG

HCN

KV10-12

KIR

The mouse and human channel sets are virtually identical

?

K2P

GLUR

Functional analysis of channels

Patch clamp recording technique What do channel currents look like?

Patch Clamp Recording Configurations

Glass micropipette connected to amplifier

Voltage clamp control voltage and measure current Used to study channel gating

Current clamp control current and measure voltage Used to study neuronal activity

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

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
1 channel

3 channels
dozens of channels hundreds of channels time (ms) I (pA)

Macroscopic voltage clamp recordings allow quick measurement of voltage-gated channel kinetics and open probability

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)

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 in excitatory channels or loss of function in inhibitory channels. Migraine, ataxia and neuropathic pain are other examples of channelopathies.