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Cell Membrane Lecture Notes 2 Notes

Pharmacology Notes > BIOL21141 Cell Membrane Structure & Function Notes

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Lecture 18 - Ion Channels
Ion Channels:
 Determine Cell Phenotype.
 Define Function.
 Have roles in Pathobiology - "Channelopathies".
 Drug Targeting and Drug Development (eg: Na-channel inhibitors: anti-arrhythmic, local anaesthetics, K-channel inhibitors/openers: insulin secretion, antihypertensive, HERG Kchannel in drug toxicology screening).
 Nature Warfare (Toxins produced by many animals).

Ion Channel Organisation - Functional Signalling Unit

For example, in the heart muscle within the ventricles.

What's very different between cardiac and skeletal muscle is the types of ion channels expressed in the cell membrane and how these are linked through cell signalling mechanisms to those common contractile processes.

Cardiac muscle has a very long action potential,
lasting around 400ms, but skeletal muscle potentials last around 5ms.

It is the type of ion channels that are expressed in the cell membrane, which determines these properties.

For example, the calcium channel facilitates calcium entry and it generates a signal not associated with contraction and instead one which is associated with other ion channels located along the sarcoplasmic reticulum. It binds to the receptors on the sarcoplasmic reticulum and these allow calcium to come out of the internal stores to act as the signal
[Calcium-induced-calcium release].

The geo-positioning of the ion channels is also really important, in the cardiac myocyte they are located in the T-tubules and this is as they have clear and short access to the ryanodine receptors on the sarcoendoplasmic reticulum store.

Channelopathies

Cystic Fibrosis is a hereditary disorder characterised by lung congestion and infection and malabsorption of nutrients by the pancreas.

Cystic Fibrosis; 1 in 25 people are carriers of CFTR mutations.

This disease is characterised by the inappropriate fluid and electrolyte transport
(Cl-) across the epithelia in the lungs, resulting in the accumulation of mucous in these structures. Drug Development

There aren't many drugs that target ion channels as they are quite difficult to target.

However, they do exist (eg: Lidocaine), but many cells have common types of ion channels,
and when they have unique actions in a cell these are hard to target specifically without producing massive side effects.

Ion Channels and Cell Viability
 Ion channels are crucial elements for the activity of all living cells

Ion concentrations are different inside vs. outside cells,
and channels and exchangers establishing these differences.

Each living cell is characterised by an electrical potential difference between the cytoplasm and the extracellular medium; which varies between -40 and -90 mV.


An electrochemical gradient across a plasma membrane exists for each ion species.
Cells use these gradients in their signalling and control systems.

Ion channels are key in the generation of the membrane potential.

What is an Ion Channel?

Plasma membranes are lipid structures, and ions cannot cross these membranes, so ion channels are needed to cross.

Organelles also have ion channels.

An Ion channel is a transmembrane protein that form a selective pore which allows ions to passively cross a cell membrane.

Ion channels contain a gate that permits (when "open") or prevents
(when "shut" or "inactivated') the passage of ions, through
"Gating".

The channels exhibit regulated and stochastic (random) gating between their open and shut states.

Ions move through favorable gradients:

Electrical.
Chemical - concentration gradient.
Electrochemical. When is a Channel a Pore?
Channels which are not gated, are referred to as
"pores" as opposed to a (gated) channels.

Pores don't have a gate and are nondiscriminating, these are much faster than carriers.

 Pore - Always Open. N/A Particles per event. 2x109 particles per second.
 Channel - Intermittently open. Unitary Opening. 6x104 particles per event. 106-108 particles per second.
 Carrier - This is never fully open. There are cycles of conformational change. 1-5 particles per event. 200-50,000 particles per second.

How Ion Channels are Regulated

Ion channels can be regulated in 3 main ways.

Ligand-Gating is one of these where a channel is activated by acetylcholine, which binds to specific sites on the membrane, once bound it causes a conformational change.

Mechanosensitive channels sense pressure changes in cells and then external environment and once mechanical pressure is felt it opens, allowing ions to cross.

Voltage-Sensitive channels open when the membrane potential changes.

Types of Ion Channels / Classification
 There are more than 300 Types of Ion
Channel.
 There are more than 500 Genes encoding Ion Channel Subunits.

Ion channels can be classified by Selectivity, Gating and their Localisation.

There are two broad groups that select for Cations (Na +, Ca2+ and K+) and those that select for Anions (Cl-), whilst some channels only select for certain ions.

What the channels are gates by: Ligand or Voltage is also very important. Ligands include an ion, a small organic molecule or protein, whilst a voltage gated channel opens in response to depolarisation and hyperpolarisation.

The localisation of the channel is also important, for example is it in the plasma membrane or on an internal organelle. Classification through Molecular Structure

Another way to classify them is by looking at their molecular structure to classify them and categorise them.

For example, Single and Four-Domain channels can be used to classify a group.

143 members of the structurally related ion channel genes

Representation of the amino acid sequence relations of the minimal pore regions of the voltage-gated ion channel superfamily.

The Generation of a Membrane Potential
 Only a small number of ions need to flow in order to establish the potential. Therefore, the concentrations of ions remain relatively unchanged.

As an electrical gradient builds up, it counteracts the concentration gradient. At equilibrium the net flow of potassium is equal to zero. This value is called the equilibrium potential.

Equilibrium Potential

The equilibrium (or Nernst) potential is the membrane potential at which there is no net flow of an ion species from one side of the membrane to the other.

 At EQ although net flow is null; ions move freely across the membrane.
 The Nerst Equation allows you to calculate the
Reversal Potential across a cell membrane. +65mV
-98mV
128mV
-91mV

The
Goldman-Hodgkin-Katz (GHK) Equation

Cell membranes are permeable to several ions.

The GHK equation is used to calculate the resting membrane potential (Em) of a cell permeable to more than one ion, when the permeability (P) of the cell to each ion is known.

The Resting Potential of most cells is close
EK

The resting membrane potential is dominated by the ionic species that has the greatest permeability across the membrane.

The fact that the resting potential is nearest to E K suggests that K+ is the ion most permeable under resting condition (ie: K+ channels are open at rest).


K-channel inhibitors - Depolarization.
K-channel activators - Repolarise/Hyperpolarisation.

Potassium Ion Channels
 Repolarization.
 Hyperpolarization.
 Resting Membrane Potential.

to the 

Potassium channels are very important for setting the resting membrane potential, controlling the repolarisation potentials and also causing hyperpolarisation of the cell membrane.
The sodium/potassium ATPase sets up a favourable gradient across the cell so that potassium ions always flow out of the cell.

Sodium Ion Channels

Sodium channels bring about different biophysical changes to the cell membrane potential,
as there is always a greater concentration of sodium outside of the cell, so whenever the channel opens there will be depolarisation of the membrane.

Calcium Ion Channels
 Depolarization.
 Elevation of Intracellular Calcium.

Calcium channels do the same thing on the plasma membrane, there are many different types, some activated at low depolarisations of the cell and others at high depolarisations of the cell.

Calcium entering the cell causes depolarisation and an elevation of intracellular calcium through calcium-induced-calcium release.

The Action Potential Profile
 Resting Membrane Potential; Open K-channels.
 Threshold Potential; Closure K-channel, Opening
Na-channels.

Not all cells generate action potentials, but all cells have a membrane potential.

 Depolarization; Open Na-channels, Open Ca-channels.
 Transition Period: Inactivated Ca/Na-channels, Open K-channels.  Repolarization; Closed Na-channels, Open Kchannels.
 Hyperpolarization; Open K-channels.
 Refractory Period; Inactivated K-channels.

Lecture 19 - Structure of Ion Channels
Experimental Methods of Studying Ion Channels
Biochemistry

Protein isolation and purification [eg: Western Blot] can be used to study Ion channels.
However, this is challenged as there often isn't an availability of high affinity ligands, as affinity chromatography cannot be performed.
Radioactive ion flux - Proteins can be reconstituted back into the lipid-bilayer (Can transfect cells and then measure their efflux for activity and electrical activity).

Molecular Biology

Ion channels can be studied using: Sequencing, Cloning, Mutagenesis.

Structural Biology

Can be studied using: Atomic force microscopy, Crystallography, Nuclear magnetic resonance.

Electrophysiology

Electrophysiology is a dynamic and versatile technique to record ion channels in living cells.

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