Membrane and Synaptic Physiology

Membrane Bioelectricity

• An ion channel is a battery (concentration gradient) in series with a resistor (pores).
  - The membrane acts as a capacitor as it can store charge and produce a voltage.
• There is an electric field between a positive (+) and negative (-) pole. There is also a voltage running from the negative to the positive pole.
  - Voltage is known as the electrical potential energy per unit charge (volts).
  - The voltage at a particular location is the net 'work done' (energy stored) moving a 'unit' of positive charge from far away, through the electric field to that location. Voltage increases as a positive charge is pushed against the field.
  
• Current = Charge / Time - The charge flowing per unit time is the rate of flow of charge, i.e. charge over time. This corresponds to current (amps).
  - In a metal wire (with 6x10^18 electrons/s) there is a sea of jiggling electrons bathing in a lattice of metal ions. Applying an electric field along the wire will move the electrons to the positive end.
  - Electrons are repelled by a negative voltage, and attracted to the positive voltage. The direction of the current is the direction of net poitive charge flow, i.e. opposite to the direction of electron travel.
  

Membrane Bioelectricity

• Ohm's Law:
  - Voltage = Current x Resistance
  - For many conductors, current is proportional to voltage drop. Current flows downhill, down a voltage gradient, from high (positive) voltage to low (negative) voltage.
  
  
• Resistance = 1/Conductance - So Current = Conductance x Voltage Drop is equivalent to Voltage Drop=Current x Resistance.
  - Conductances in parallel add.
  
  
• Capacitors: - Their basic structure is a 3 layered sandwich of two conducting plates on the outside (of metal or ionic solution) and an insulator in the middle (e.g. air or a lipid).
  - A dielectric consists of polarisable molecules, i.e. an electric field can move charge from one end of each molecule to the other, and/or line up the molecules so their internal electric fields cancel out much of the overall electric field within the dielectric, allowing a greater voltage to be placed across it before the insulation breaks down.
  

Membrane Bioelectricity

• Capacitors:
  - Charge is stored and there is a voltage across the places.
  - The Charge stored = Capacitance x Voltage.
  
  

  - In a charged biophospholipid bilayer membrane, there are excess negative ions on one side and excess positive ions on the other. The opposite charges are electrostatically attracted to one another via the lipid but they cannot pass through, so they are 'held' loosely on opposite faces of the membrane.

  - Capacitance allows membranes to add up (integrate) inputs.
  Membrane capacitance (F) adds up currents over space and time and produces the membrane potential. If a capacitor is storing charge (Q) then the voltage across it is Q/C.
  - Capacitance is also known as permittivity x area/thickness.

  - The capacitor accumulates (integrates) current flowing onto one plate and off another as charge is stored, i.e. when current flows through a capacitor it just flows onto one plate and off another. No actual current physically crosses the gap between the plates, instead the charge on the plates go up on one and down on the other.

Membrane Bioelectricity

• Capacitors:
  - The electric field = Charge per Area / Permittivity.
  - Voltage = -field x distance.
  
• Membrane Integration Cycle:

Classification of Transporters

The plasma membrane separates what is inside the cells from the outside. In nerve cells, their membrane composition of lipid/protein is 4:1. Whereas, in a mitochondrial inner membrane it is 1:4. Red blood cells have more protein than lipid.

The phospholipid bilayer is a basic structural unit of membrane containing amphipatic phospholipid molecules. The bilayer can be penetrated only by small and lipid molecules (i.e. uncharged) e.g. CO2, urea, etc.

Proteins function to connect two phases:
- Extrinsic or peripheral membrane proteins.
- Intrinsic or integral membrane proteins.
Intrinsic membrane proteins can span the membrane one or more times:
- They need a sequence of >3nm (20+/- amino acids).

Kyte-Doolittle Hydropathy Plot: - On the x axis is amino acids; on the y axis is hydrophilicity.
- The running average finds which amino acids are most hydrophobic to see which line the membrane, i.e. predict the membrane spanning alpha-helical rods.

Classifications of Transporters

Hormone and neurotransmitter receptors have 7 transmembrane domains, whereas transporters have 12.
Many transport proteins have hydrophilic N and C terminal tails.
- Glycosylated amino acids are found in extracellular domains. These help fix the protein in the cell membrane (targetting) and helps orientation.

Intracellular sequences:
- Are targets for chemical modification (phosphorylation).
- Interaction regulatory molecules (e.g. calmodulin) so they can change the kinetics.

Lipids and proteins are undergoing fast rotational movements:
- Flip flop (is energetically unfavourable)
- Therefore lateral diffusion is more common as it is favourable.

Proteins move slowly; even slow when coupled to the cytoskeleton.

Protein-mediated Solute Transport: Characteristics to distinguish from simple diffusion:
1) Saturation - A finite number of transporters show saturation and channels show less.
- Enzyme kinetics, i.e. increasing solute concentration means more is transported.

2) Selectivity - Transport exclusively or preferably certain compounds or ions.
- Channels tend to show lower specificity.

3) Competition - Accounts for less than absolute specificity; Similar molecules use the same pathway and slow eachothers permeation; Similar solutes compete for the same binding sites.
- In certain conditions, Calcium ions can be made to permeate Sodium channels.

4) Pharmacological Inhibition - Used to characterize the transporter. Diuretic amiloride inhibits the renal Na+/H+ exchanger; TTX (tetrodotoxin) blocks sodium channels; Curare blocks ACh.

5) Asymmetry - Is a unique property of protein mediated transport. In channels it is termed rectification where an ion prefers to pass in one direction than another first.
- In transporters such as the Na+/H+ exchanger the rate of exchange differs in external vs internal pH. In the Glucose carrier, the Vmax of Gluc influx is 10 times greater than efflux.

Classification of Transporters

• Classifying Transport Phenomena:1) Energetic
  - Active transport is when the solute is transported against the electrochemical gradient.
  - Primary active transport is directly coupled to ATP hydrolysis. (e.g. pumps/ATPases such as calcium, Na+/K+, proton).
  - Secondary active transport is the solute gradient used to fuel the movement of other solutes. (e.g. the Na+ gradient generated by the Na+/K+ pump; or the H+ gradient by the H+/K+ pump).
  
  

  2) Mechanistic (the use of solute coupling)
  - Uniports have one directional movement down a chemical gradient. They have single, non coupled fluxes.
  - Cotransport or symports involves the substrate being coupled to movement of other substances in the same direction.
  - Exchange or antiports involved the movement of substrates linked to the opposite movement of its partner.
  

Classification of Transporters

3) Net Charge Translocation
- Electroneutral = no net charge occurs during transport.
- Electrogenic/Rheogenic = Net charge translocation occurs, i.e. due to the inbalance of 3Na+ out and 2K+ in for neurones, or 1Ca2+ out and 3Na+ in.

• Uniporters:
  - Have the best understood mechanism of facilitated diffusion, for example, of glucose.
  - GLUT1 to GLUT5 are uniporters with different affinities for glucose, 500 amino acids and 12 transmembrane segments.
  - They are expressed on cell surfaces and intracellular vesicles. The GLUT transporters can be regulated via phosphorylation.
  - Normal plasma glucose levels are between 3.5mM and 10mM.
  - GLUT3 has a Km value of 1.8mM and GLUT2 has 13.2mM.
  - The brain glucose transporter (GLUT3) works close to maximal velocity and the livers uptake of glucose is controlled by prevailing glucose levels (GLUT2).
  - Transactivation of glucose uptake: the presence of glucose at the other side facilitates the entry of glucose.

Classification of Transporters

• Antiporters:- Act as secondary active transport systems. They act to rapidly exchange products of enzyme reactions with their substances.
  
  

  - The Na+/H+ exchanger regulates intracellular pH, cell volume and Na+ transport.
  (NHE1 to NHE6; two functional clusters where the N terminal mediates exchange or the C terminal is the regulatory domain).

  - The Cl-/HCO3- exchangers (AE1 to AE3):
  AE1 is also known as band 3 protein which has a role in blood CO2 transport and a structural role; whereas AE2 protects against alkalinization.

  - NHE1 and AE2 maintain the basal pH and also play a role in volume regulation.
  Cell shrinkage activates NHE1 which causes alkalinization. This activates AE2.
  The net result is Na+ and Cl- entry and thus H2O entry.

Semi-Permeable Membranes and Resting Membrane Potential

• Semi-Permeable Membranes:The key components of a semi permeable membrane include:
  1) A lipid bilayer surrounding the cells which is impermeable to charged ions (hydrophilic). This acts as a capacitor and stores charge near the surface of the membrane on either side to generate membrane potential between separated unlike charges.
  
  

  - However, ions can cross the membrane via:
  2) Pump proteins (and other transporters) which consume metabolic energy to set up ion concentration gradients, undergo slow pumping mainly by the Na+/K+ ATPase (against concentration gradients) and stores energy in concentration gradients.

  3) Ion channel proteins (each represented by a 'battery' and its internal conductance) which can pass ions much faster, rapidly harnessing energy stored in concentration gradients to send signals, e.g. to change the voltage across the membrane or intracellular calcium.

Semi-Permeable Membranes and Resting Membrane Potential

• Electrophysiological Recording Techniques:Extracellular Recording (metal, hollow saline-filled glass or silicon electrode)
  - Records action potentials etc from a single cell or group via placing the electrode on brain tissue.
  - Recordings are upside down.
  - Set up includes a 'faraday cage' which shields out non biological noise, and a pre-amplifier which allows modification of the signal.
  
  

  Intracellular Recording (sharp electrode with an electrolyte filled hollow glass 'spear')
  - Records the membrane potential changes (first founded by Hodgkin and Huxley.

  Patch Clamp: Is used to measure whole cell currents. The patch electrode tip is ~1-2um so it can be used for both voltage recording and current injections as there is low resistance access. It is less damaging so can use less robust/smaller cells.
  - The perforated patch technique is when an antibiotic pokes holes in the cell membrane to keep it metabolically intact.

Semi-Permeable Membranes and Resting Membrane Potential

• Electrophysiological Recording Techniques:
  Voltage Recordings - Record the voltage +/- injecting a constant current +/- defined pulses or waveforms of current into the cell/ fix the current (i.e. 'current clamp') and allow the membrane potential to do its own thing.
  

  Current Recordings - Control voltage with a voltage clamp with an electrode tip diameter of 0.1mm (i.e. hold at a constant voltage or impose voltage pulses) and thus take control of the membrane potential.
  - Compare the actual current to the desired current; if the actual current is too negative then less voltage should be injected and vice versa.
  - Inward currents that carry net positive charge into a cell are nearly always shown as negative/downwards.

  In practice, to hold the membrane potential constant the experimenter often injects an equal and opposite current to the one being injected by the ion channel to cancel it out.
  - No net current means no net charge into the cell so there is no voltage change. So if the biological current is Ibio, the experimenter injected 'clamp current' (Iclamp) is -Ibio.

Semi-Permeable Membranes and Resting Membrane Potential

• Electrophysiological Recording Techniques:- Impaling a cell with a sharp electrode: the membrane could seal onto the outside of the glass, it may be leaky. The electrode has a small tip so it has high resistance, is noisy, and has limited current passing or voltage control.
  - Whereas, sucking a patch of cleaned membrane into a clean pipette so it sticks onto the inside of the glass (very tight seals possible, as well as lower pipette resistance, better current passing, voltage control and lower noise).
  
  

  Patch Recordings
  The Giga seal (>10ohms) ensures negligible current leaks out under the rim of the pipette. This reduces noise ad enables the detection of very small currents (pA) coming from single channels.

  Patch configurations:
  - Cell attached, is when if a patch of membrane is ruptured without damaging the cell a macroscopic recording using intracellular solutions can be made. So there is an inside-out patch recording.

Semi-Permeable Membranes and Resting Membrane Potential

Patch configurations:
- Whole cell recording is when there is a strong pulse of suction so there is a rupture of the membrane and the cytoplasm is continuous with the pipette inferior. When the pipette is retracted, a membrane and cytoplasmic tube forms which stretches and thins so that it ruptures from the rest of the membrane. Then, the ends of the membrane re-anneal on the outside of the pipette to make the extracellular domain accessible.
Advantages of this is that it allows the Vm to be controlled and the outer face of the membrane can be accessed while the external solutions can be controlled. Thus it gives access to receptors.
Disadvantages of this include the cytoplasmic factors are lost and it is not physiological.

- With single channel currents where the patch clamp configuration is outside-out, there are stochastic (random) openings and the open probability = the fraction of time that the channel is open.

Average Conductance = Open Conductance x Po

Semi-Permeable Membranes and Resting Membrane Potential

• Membrane Potential:- Membrane potential (Voltage) is always given by Charge Stored / Capacitance.
  Lipid membranes have a capacitance which stores charge, producing a voltage across the membrane.
  Charge = Current x Time
  
  

  Batteries:
  - Biological membranes can be 'energised' by energy-consuming ion transport proteins pumping ions across them, setting up a concentration gradient.

  - An ion concentration gradient is a battery. The ions tend to diffuse down their concentration gradient, purely by random motion, and hence can drive electric currents in that direction if they are positive, or in the opposite direction if they are negative.

  - The bigger the concentration gradient, the more energy stored per ion, and per unit charge of ions and hence the bigger the voltage. When batteries are in series the voltages add.

Semi-Permeable Membranes and Resting Membrane Potential

• Membrane Potential:Ion Channels
  - A transmembrane ion channel (pore) protein behaves as a conductance in series with a battery, whose voltage depends on the concentration gradients of the ions that can pass through the channel.
  - Voltage is the electrical potential energy per unit charge. The ion concentration gradient plus a selectivity filter acts as a battery, converting chemical energy into electrical energy.
  - The effective voltage between battery 'terminals' is how much energy is released, per coloumb of charge, when ions move down their concentration gradient or how much energy must be put in to move them up their concentration gradient.
  
  

  - In the absence of a membrane potential (Vm=0) the current in the resistor flows in the direction of voltage drop, from high to low voltage (i.e. from more positive to more negative).

  - Vx, where x is a channel or ion type means the reversal, battery or equilibrium potential and is used synonymously with Ex or Erev. When a channel is selectively permeable to only one kind of ion, its battery voltage is well approximated by the Nernst equation:
  Erev = 60ln([X]out // [X]in)

Semi-Permeable Membranes and Resting Membrane Potential

• Ion Channel Structure:
  - The selectivity filters of most cation channels are found near the extracellular opening.
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  - In a voltage gated K+ channel, the narrow part of the pore region is found embedded in the membrane. Batteries and capacitors are connected in series and the voltages across them add.
  - When there is a transmembrane potential, the effective voltage drops down the membrane potential as ions go through the channel conductance.
  
  

  The electrochemical gradient, or Driving Voltage (Vdrive) is calculated by Erev - Vm.
  - The channel acts as a charge injection device; where the current injected into a cell is the conductance x Vdrive.

Semi-Permeable Membranes and Resting Membrane Potential

All channels draw the membrane potential closer to their Erev:
- K+ currents hyperpolarise membrane potential (make it more negative) as they are inhibitory.
- Na+ currents depolarise the membrane potential (make it more positive) as they are excitatory.
- Cl- currents can do either depending on the Ecl and Vm.

• I-V Curves:
  - Under voltage recording vs voltage clamp.
  With an inward current (current entering the cell via channels of type x)

Semi-Permeable Membranes and Resting Membrane Potential

Outward current (Ibiological) (Current leaving the cell via type x channels)

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Potassium leak conductance:
- Much of this is from non specific cation channels, which behave like small holes in the membrane that inject positive current whenever Vm is negative, depolarising the membrane above Ek.

Semi-Permeable Membranes and Resting Membrane Potentials

Chloride channels are hyperpolarising:

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- Membrane potential is driven towards the conductances reversal potential. If Vm is above (positive to) Erev, the injected current is hyperpolarising whereas if Vm  is below (negative to) Erev the injected current is depolarising.
- When there is zero net current flowing onto the membrane capacitance, we get a steady state membrane potential.
Vm = fkEk + fnaEna where fk = gk/gtotal.

Semi-Permeable Membranes and Resting Membrane Potential

Reversal potentials of channels permeable to multiple ion species can also be approximated by the average of Nernst potentials, weighted by relative conductances:

Erev = gnaEna + gkEk + gclEcl / (gna + gk + gcl)
It can sometimes be more accurately predicted by the GHK voltage equation.
Reversal potentials are often just measured empirically.

For channels with multiple ion permeabilities or 'speeds' of movement through channel, a weighted version of the Nernst equation can also be used to calculate reversal potentials:
Erev = RT/zF ln (pK[K+]o + pNa[Na+]o + pCl[Cl-]i // pK[K+]i + pNa[Na+]i + pCl[Cl-]o)

• Resting Membrane Potential:- The membrane has diffrent permeabilities to different ions and the GHK equation takes this permeability into account.

Action Potential Generation

1) Action potentials are a digital-like output pulse of neurones and other cells, leading to a biological action.

2) Threshold, 'all-or-none', if Vm is even slightly below threshold it will decay back to the resting level. If Vm is slightly above the threshold there is a much larger response at approx. 100mv x 1-2 pulses.

3) 'All-or-none' is stereotyped, but not identical.
- Action potentials can vary by >2-fold in height and duration, so threshold can vary.
- e.g. Relative refractory period, inhibitory inputs and neuromodulation.

4) Key ion channels: Depolarisation-activated (voltage dependent) Na+ and K+ channels.

Action Potential Generation

• Sequence of Events:  - Depolarisation rapidly opens Na+ channels to cause a more inward current which charges the membrane capacitance and thus there is more depolarisation leading to the rising phase (positive feedback loop) i.e. action potential peak. The depolarised Na+ channels close or inactivate again.
  
  

  - Depolarisation opens K+ channels more slowly causing an outward current so there is a negative charge left on the intracellular face of the membrane capacitance. This causes the Vm to repolarise leading to the falling phase and then it hyperpolarises.

  - The refractory period/AHP (after hyperpolarizaion) is when K+ channels gradually shut and Vm returns to the resting membrane potential to allow the Na+ channels to recover. The neuron is initially unable to fire another action potential (absolute) then it can fire small action potentials with a higher threshold (relative). As Vm approaches resting level and Na+ channels recover, threshold falls and action potential size recovers.
  

Action Potential Generation

• Anatomy of an Action Potential:
  

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- Myelinated axons speed up action potential propagation via saltatory conduction as depolarisation only takes place at the nodes of Ranvier rather than the whole length of the axon.
- Membrane conductance increases during action potentials (channels open).

Action Potential Generation

• Voltage Clamps:- In practice, to hold the membrane potential constant the experimenter often injects an equal and opposite current to the one being injected by the ion channels to cancel it out.
  - No net current means no charge into the cell so there is no voltage change. Thus, if the biological current is Ibio, the experimenter-injected current Iclamp = Ibio.
  
  
• Selective Ion Channel Blockers:
  - Tetrodotoxin (TTX) is a toxin produced by puffer fish and blue-ringed octopuses (synthesised by symbiotic bacteria). It specifically blocks Na+ channels so action potentials cannot be generated.
  - Explaining Current-Voltage relationships:
  The slope of the I-V relation corresponds to conductance (V=IR) so I=gV.
  An increasing slope means an increasing conductance, thus more channels opening.
  Conductances in parallel add.
  The Vdrop across the resistors can be thought of as 'E' or it can be membrane potential.
  

Action Potential Generation

Two conductances with different reversal battery potentials (Erev): a positive Erev means an outward current and a negative Erev means an inward current.

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- If 2 voltage dependent conductances with different Erevs open sharply at the same Vm, they have the same starting current.

Action Potential Generation

• During Action Potentials:- During an action potential, the sodium channels rapidly open and potassium channels open more slowly to produce a graph like this:
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  - Positive feedback loop: Depolarized membrane potential opens Na+ channels which increases the Na+ current to depolarize the membrane more.
  - Negative feedback loop: The depolarized membrane potential opens K+ channels to increase the K+ current which stops the depolarization of the membrane potential.
  
  

  - Permeability changes: During an action potential the permeability of the membrane to Na+ increases by 600x compared to resting Vm. To K+ it is 300x more permeable.
  

Action Potential Generation

Conductance changes:
- An action potential is bounded by a region bordered on one extreme by the Potassium equilibrium potential (Ek) of -75mV, and on the other extreme by the Sodium equilibrium potential (Ena) of +55mV.
- The resting Vm is -60mv. It is not equal to Ek because there is a small resting Sodium permeability maintained by the Na+/K+ ATPase pump.

Hodgkin & Huxley used a voltage clamp to increase the membrane potential at certain points or levels and measured changes in Na+ conductances. The more the cell is depolarized, the greater the conductance. This is similar to K+ conductances.

Currents:
Ii (net ionic current) = Ina - Ik
- This is what charges the membrane capacitance. So the rate of change in Vm tracks Ii.
IR = Vm - Vrest.
The rate of voltage change = the change in voltage / change in time (dV/dt)
Ii = C(dV/dt)

Action Potential Propagation

• Sequence of Events:1) The mechanism of action potential generation at a single site.
  
  

  2) Voltage difference between the action potential site and the next segment of axon drives positive current axially along the tube of the cytoplasm. Iaxial = (V1 - V2)/Raxial.

  3) This axial current deposits positive charge on the intracellular surface of the membrane capacitance of the next segment of the axon, which repels positive charge form the extracellular surface making the membrane potential less negative.

  4) This depolarization causes Na+ channels to open so that more positive current enters the axon.

  5) Axial and sodium currents causes more depolarization which crosses threshold so there is positive feedback at a local patch of membrane, and thus the action potential will reproduce itself.
  

Action Potential Propagation

• Sequence of Events:
  6) The refractory period prevents the re-excitation of the zone that the action potential has just passed.
  
  

  7) Myelin increases the thickness of the membrane which reduces capacitance and the amount of charge needed to depolarise the membrane. This results in a faster propagation.

• All-or-None Responses:- Responses at different stimulus strengths are all or none but not identical. Spike waveform and threshold vary during repetitive firing. The second action potential has a higher threshold with a shorter, wider spike.
  - The origin of the action potential threshold: Bistable instantaneous net I-V relation: When there is sufficient depolarisation activated Na+ channels in the membrane, there is positive feedback between the voltage and current. A positive change in voltage leads to a more positive current which leads to a more positive voltage etc.
  - There is no action potential possible when most of the sodium channels have inactivated. If some of them are inactivated then the membrane can still be bistable but witha higher threshold and lower up-state (so a smaller action potential).
  

Action Potential Propagation

• Action Potential Initiation:
  At a typical axon initial segment:
  - Most action potentials are initiated in the axons. The 'axon initial segment' fires first even when the current is injected into the soma. This is due to a higher sodium channel density, and because sodium channels open at more negative membrane potentials than the soma-dendritic sodium channels.
  - An axon 'bleb' is when an axon is cut and the membrane reseals. When current is injected, the action potential then propagates into the soma.
  
  
• Unmyelinated Axons:- Forcing positive charge onto the intracellular surface of the membrane capacitance depolarises membrane potential.
  1) Push the positive current onto the upper surface of the bottom plate for time t.
  2) Charge accumulates at the upper surface of the bottom plate and repels the same amount of positive charge off the top plate.
  3) A negative charge is left behind on the lower surface of the top plate. Positive current is repelled off the top plate over time t.
  

Action Potential Propagation

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Connecting neighbouring segments of an axonal or dendritic cable:
- Current (charge) can flow along the cytoplasm, if the neighbouring compartments are at different voltages. If membrane charge spreads, membrane voltage spreads with it.

Action Potential Propagation

• Axial Charge Flow:- Axial current (Iaxial) flowing into the nth compartment from one of its neighbours is calculated as Voltage Drop / Raxial, or Iaxial = gaxial x Vdrop.
  (Raxial is the axial cytoplasmic resistance from one compartment centre to the next).
  - Iaxial (from compartment 1 to 2) = gaxial (V1-V2). For each compartment Itotal = Iax + Ichannel
  
  

  An action potential propagating down an unmyelinated axon:
  1) Sodium channels open in response to a stimulus, generating an action potential at one end of the axon. Some depolarising current passively flows down the axon.
  2) Local depolarization causes neighbouring Na+ channels to open and generates an action potential further along the axon.
  3) Upstream Na+ channels inactivate, while K+ channels open. Membrane potential repolarizes and the axon is refractory here.
  4) This process is repeated, propagating the action potential along the axon.
  - The speed of propagation is roughly proportional to the square root of the axon diameter.

  - In myelinated axons action potentials occur at the nodes of Ranvier where Na+ channels are found in the centre. K+ channels are found in the myelin sheath.
  

Sodium and Calcium Channels

• Functions:Calcium channels - Are involved in muscle contraction (e.g. L-type are involved in producing cardiac action potentials via excitation-contraction coupling)
  - Hormone and neurotransmitter secretion (movement of vesicles in presynaptic membrane)
  - Neuronal excitability.
  - Gene expression.
  
  

  Sodium channels - Are involved in nerve conduction and muscle contraction.

• Calcium Channels:- The intracellular calcium concentration (10-7 mM) is much lower than the extracellular concentration (1-2mM). A transient increase in intracellular calcium increase is caused by entry through calcium channels and acts as a second messenger coupling receptor activation to many other processes.
  - Experimental evidence using voltage clamp or patch clamps show there are different types:
  (A) Injecting EGTA into a plysia neurone, which binds to calcium to lower its concentration, causes the neurone to have a fast inward current and slower inactivation.
  

Sodium and Calcium Channels

(B) Using Barium as a calcium substitute:
- There is a larger inward current as calcium channels are more permeable to Barium, however there is a slower inactivation due to replacing calcium with barium.

• Calcium Channel Subunits:
  - The alpha 1 subunit in calcium channels is very similar to the sodium channel alpha. Voltage-dependent calcium channels exist as a complex of subunits including a1, a2, b, y and d. Each of these are units that affect the kinetics of the channel.

  - A motif of 300-400 amino acids is repeated 4 times, each peak having 6 transmembrane domains. a1 has the most of the functional properties.

Sodium and Calcium Channels

• Calcium Channel Subunits:
  Calcium channels are all interrelated:
  - L type open slow and slowly inactivate.
  - P/Q, N and R type are intermediate.
  - T type open in response to a small depolarisation and inactivate rapidly.
  - They are all modulated by the subunits. (e.g. a2 causes an increase in current and more rapid inactivation).
  
  
• Calcium Channel Types:L-Type - Are found in virtually all excitable and many non-excitable cells.
  - Have a high conductance (~20pS)
  - Are involved in EC-coupling in heart and smooth muscle.
  - Are activated by strong depolarisation, and have slow inactivation.
  - They are also blocked by dihydropyridines (DHPs) and verapamil which bind to the channel which inhibits Calcium conductance in the heart and peripherally.
  - Adrenaline gives way to more calcium in the heart.
  

Sodium and Calcium Channels

• Calcium Channel Types:T-Type - Are widely distributed in excitable and non-excitable cells.
  - They have a low conductance (~10pS)
  - Are activated by small depolarisation and have rapid inactivation.
  - They are involved in rhythmic action/pacemaker potentials in the SAN of cardiac muscle and neurones to modulate breathing.
  - They produce burst firing modes of action potentials, which are enhanced by hyperpolarisation (removing the inactivation of channels) and regulate intracellular calcium concentrations.
  - They have no selective blockers but they block Ni2+ and ethosuximide.
  
  

  N-Type - Is a high voltage activated channel found in neurones.
  - Has characteristics in between L and T type channels with a conductance of ~15pS.
  - They are found in presynaptic membranes and control transmitter vesicle release, particularly to control the sensation of pain.
  - They are blocked by omega-conotoxin which is isolated from snails.
  

Sodium and Calcium Channels

• Calcium Channel Types:P/Q-Type - Are found in cerebellar Purkinje cells.
  - Have slower inactivation compared to N type channels.
  - Conductances similar to N type (~15pS)
  - Involved in synaptic plasticity and also controls transmitter release.
  - Blocked by omega-agatoxin isolated from spiders, and polyamine (FTX).
  
  
• Sodium Channels:
  - Are composed of a, B1 and B2 subunits. They are evolved from calcium channels.
  - Skeletal and cardiac muscle have no B2 subunits.
  - SCN1B codes for the B subunit which enhances the sodium current amplitude and modifies its properties.
  - The intracellular domain has an inactivation gate (h gate).
  - The 4th domain is the voltage sensor which allows the opening of the channel; the large intracellular loop is where modulation occurs allowing phosphorylation with a protein kinase.

Sodium and Calcium Channels

• Sodium Channels:- They are responsible for the rapid upstroke (rising phase) of the nerve and muscle action potential, and they are very voltage sensitive.
  - They are blocked by Tetrodotoxin (TTX), saxitoxin (STX) and scorpion toxin (ScTx).
  
  

  - They are encoded by the SCN genes.
  SCN1A, 2A, 3A and 8A (Nav1.1, 1.2, 1.3, 1.6) are all expressed in the CNS.
  SCN4A  (Nav1.4) is expressed in skeletal muscle.
  SCN5A (Nav1.5) is expressed in the heart. Brugada syndrome is due to a mutant of this type.
  SCN9A, 10A, 11A (Nav1.7, 1.8, 1.9) are expressed in the peripheral nervous system.

  - Different genes code for channels with slightly different properties.
  - The P loops give rise to sodium ion selectivity.
  

Potassium Channels

• Ionic Gradients:The inside and outside concentrations are different due to pumps (ATPases) which set up opposing gradients. The intracellular concentration of Na+ is 15mM, whereas extracellular concentration is 145mM.
  The intracellular concentration of K+ is 120mM whereas extracellularly it is 4.5mM.
  The intracellular concentration of Cl- is 20mM whereas extracellularly it is 117mM.
  The intracellular concentration of Ca2+ is 0.0001mM whereas extracellular is 1.25mM.
  
  
• Resting Membrane Potential:

- At rest, the membrane is selectively permeable to K+ions due to the 'leak' potassium channels. The sodium, chlorine and calcium channels are all closed at rest. The pump is also pumping 3Na+ out and 2K+ in.

The net movement of potassium ions out of the cells through these channels continues until the resultant voltage on the inside becomes so negative that net movement out becomes impossible. (Intracellular voltage is always negative due to constant removal of positive ions). There is a gradient set up for K+ ions to mve out as long as there is a selectively permeable pathway.

Potassium Channels

• Resting Membrane Potential:- The potential at which there is zero net movement of potassium ions out of the membrane is known as Ek. This is very similar to resting potential in most cells, showing that resting potential is established by K+ efflux through K+ channels.
  - Ek can be calculated using the Nernst equation:
  Ek = RT/zF ln ([K+]in/[K+]out)
  
  
• Patch Clamp:- The I-V curve provides a fingerprint of the ion channel or current.
  - Using a patch clamp technique allows any movement to occur only within the glass pipette to measure the activity. The movement of K+ ions will occur through a single channel.
  - When you pop the cell it allows access to the inside (cytoplasm).
  - There is no current at equilibrium (Ek), when there is hyperpolarisation the membrane potential is more negative so there is an inward potassium current due to the negative voltage inside the cell dragging K+ ions in.
  - Depolarization is when the membrane potential is more positive so potassium ions are sent away so there is an outward potassium current.
  

Potassium Channels

• K2P Channels:- Are leak potassium channels which determine the membrane potential and have two pore domains in each subunit.
  - Two of these subunits are required to make a functional K2P channel. They are open at, and contribute to, Vm.
  - An amino acid polypeptide chain is threaded through the domains, and the N and C terminus are located inside the cell.
  - There are re-enterant loops (P1 and P2) located between S1 and S2, S3 and S4.
  - They do not rectify.
  
  
• KIR Channels:- These are inward rectifying channels. Rectification is a property of an ion channel which allows the current to move in one direction better than it moves in the other.
  - Thus, inward rectification is when current moves inwards better than it moves outwards. So the KIR channel allows the movement of K+ ions into the cell more efficiently than it allows them out.
  - They have 2 membrane spanning domains and 4 of these pore domains are required to make a functional KIR channel. They are open at, and contribute to, Vm.
  

Potassium Channels

• KIR Channels: - A whole cell I-V curve of K+ current through KIR channels shows the inward rectification. At Ek, when there is hyperpolarisation of the membrane there is an inward K+ current (shown as a downwards slope). Whereas if there is depolarisation there is a small or no current.
  - If you excise the patch of membrane and put 140mM of K+ ions on each side the Ek is 0mV.
  - Single channel I-V curves of K+ current through KIR channels shows inward rectification too. However, inward rectification is not an innate property of KIR, it is due to a Mg2+ blockage at an intracellular binding domain, this blocks the outward current.
  
  
• Kv Channels:

Potassium Channels

• Kv Channels:
  Delayed Outward Rectifying - Single channel currents show they provide repolarizing drive and do not inactivate much.
  Transient Outward Rectifying - Whole cell currents show they provide modulation of the neuronal firing pattern. The channel activates then inactivates very rapidly.
  
  
• BKCa Channels:- Are Calcium dependent Potassium channels with 11 transmembrane domains. 4 of these subunits are required to make a functional channel.
  - Calcium regulates their voltage activation, and the positive charges in S4 act as the voltage sensor. They provide burst termination in active neurones.
  

Active Transport Mechanisms and Pumps

• Pumps:
  - Can be transmembrane proteins which move ions across the membrane using energy, i.e. active ion pumps known as ATPases.
  - Can also be transmembrane proteins which move other hydrophobic compounds across the membrane, i.e. the ABC superfamily (also known as the multidrug transporters).
  
  
• P-Type ATPases:- This is a large group of tetramer pumps which have 10 transmembrane domains (70-150kDa)
  - They have 1-2 catalytic alpha subunits which have ATP binding domains, and 1-2 beta subunits which transport the alpha unit to the membrane.
  - They are inhibited by vanadate (a mimic of ATP which is non selective).
  - Includes Na+/K+ ATPase which controls the resting membrane potential of neurones,
  Calcium ATPases which are involved in the contraction of muscle.
  H+/K+ ATPase.
  

Active Transport Mechanisms and Pumps

• Na+/K+ ATPase:
  - Its function is recognised by 3 key observations: The role of ATP (adding ATP allows the generation of a Na+ gradient across vesicle membranes); ATPase activity (particular proteins are involved in transporting); Ouabain inhibition (extracted from Strophanthus seeds and some relatives are also expressed in the skin of toads).
  - It has a tetramer structure with alpha2 subunits similar to the calcium ATPase; and a beta2 subunit polypeptide which has a regulatory role in folding the alpha subunit in the endoplasmic reticulum to allow the transfer and insertion into the plasma membrane.
  - It has a large intracellular loop with an ATP subunit.
  
  

  1) 3 Na+ ions bind to the high affinity Na+ binding site on the intracellular surface.
  2) ATP binds and is converted into ADP, and aspartate is phosphorylated.
  3) Causing a conformational change(E1 to E2) in the pump to so Na+ transport is outward.
  4) The Na+ ions are dissociated from the pump due to a change of the binding sites to a low affinity, and the K+ binding site becomes high affinity so K+ ions bind.
  5) This results in the hydrolysis of aspartyl phosphate to change the conformation of the pump back to E1 exposing the K+ binding site to the intracellular face where the it has a low affinity so K+ ions dissociate.
  

Active Transport Mechanisms and Pumps

• Calcium ATPases:
  - There are 3 types of Calcium ATPases: Plasma Membrane (PMCA) which transport calcium ions out of the cell; Sarcoplasmic Reticulum (SERCA) which transport calcium ions from the cytosol into the sarcoplasmic reticulum; and Golgi Membrane (SPCA) which transport calcium ions and manganese ions into the golgi.
  
  

  PMCA:
  1 - Found in all tissues; 2 - Found in the brain and excitable tissues such as the heart and CNS; 3 - Found in the brain and excitable tissues including skeletal muscle and the CNS; 4 - Wide dist.
  - Each alpha subunit transports 1Ca ion per ATP hydrolysed.
  - It has an electroneutral mechanism where one calcium ion is exchanged with 2H+ ions.
  - Inhibited by lanthanum (La3+) which is non selective, or Eosin (only binds to intracellular face)
  - Calmodulin regulation: Calcium binds and activates calmodulin which causes an allosteric activation of Calcium ATPase.
  

Active Transport Mechanisms and Pumps

• Calcium ATPases:
  SERCA:
  1 - Found in fast twitch skeletal muscle; 2 - type a are found in slow skeletal muscle, type b are found in general and smooth muscle; 3 - Have a selective expression in non muscle e.g. lungs and GI tract.
  - The alpha subunit transports 2 Calcium ions per ATP hydrolysed.
  - It is inhibited (slowed down) by phospholamban.
  1) 2 Calcium ions are bound to high affinity calcium sites on the cytoplasmic face and ATP binds.
  2) ATP phosphorylates the aspartate residue to produce ADP and cause a conformational change in the pump from E1 to E2.
  3) This generates two luminal low Calcium binding affinity sites to dissociate calcium ions from the exoplasmic surface into the SR.
  - Calcium ATPases have 10 transmembrane spanning alpha helices; 4 identified calcium ion binding sites on these TM 4, 5, 6 and 8; and a cytosolic domain determined by trypsin digestion.
  - In PMCA the low affinity TM 5 binding site is missing.
  

Active Transport Mechanisms and Pumps

• Physiological Regulation Example:
  - Phospholamban inhibits SERCA's ability to take up Calcium ions. Phosphorylation acts to accelerate relaxation during sympathetic stimulation. If there is more calcium ions for contraction and less time for relaxation the rate of uptake should increase, however unphosphorylated phospholamban slows the Calcium ion uptake. Phosphorylating phospholamban removes the inhibition.
  
  

  - Phospholemman accelerates the Na+ pump so it is more effective in removing excess Na+ ions. Phosphorylation of phospholemman acts to limit the rise in intracellular sodium ions during sympathetic stimulation. When there is more Na+ entry then the ions have to be removed during dialysis.
  

Active Transport Mechanisms and Pumps

• V-Type ATPases:
  - Generally consists of 3 kinds of transmembrane proteins and 5 kinds of extrinsic polypeptides.
  - They transport only H+ ions and do not involve a phosphoprotein intermediate.
  - Found intracellularly in plant vacuoles or lysosomes and use energy released by ATP hydrolysis.
  - They function to acidify the lumen of organelles and can also be found physiologically in the plasma membrane, e.g. in Osteoclasts (important for bone remodelling/resorption).
  - Requires Cl- channels for the creation of the H+ gradient.
  
  

  - Contains 2 discrete domains: the cytosolic hydrophilic domain (V1) where A and B subunits contain sites for ATP, and helps to propel protons through V0;
  and the transmembrane domain (V0) where c and a subunits form a proton conducting channel.
  

Active Transport Mechanisms and Pumps

• F-Type ATPases:- Also consist of 3 kinds transmembrane proteins and 5 kinds of extrinsic polypeptides, and transport only H+ ions.
  - Are found in the mitochondria and power the synthesis of ATP from ADP and Pi using the H+ gradient.
  - Consists of two domains: F1 the soluble domain, and F0 the membrane domain.
  - H+ movement is converted to torque to drive ATP synthesis.
  - 4 H+ ions pass through the channel to form 1 ATP molecule, and as they move through they generate torque to allow the gamma subunit to rotate which joins ADP + Pi to form ATP.
  
  
• ABC Superfamily:- The ATP binding cassette superfamily has 12 transmembrane domains and there are a variety of subfamilies (ABCA - ABCG).
  - Each transporter tends to be specific for a single substrate, e.g. sugars, amino acids, cholesterols, bile acids, phospholipids, peptides, proteins and toxins.
  

Active Transport Mechanisms and Pumps

• ABC Superfamily:- There are 4 'core' domains: 2 Transmembrane domains (the passageway); and 2 Cytosolic domains (ATP binding domains).
  - Multidrug resistance of tumour cells against, e.g. microtubule inhibitors colchicine and vinblastine, which is due to overexpression of MDR1 (ABCB1), a 170kDa glycoprotein. Transported substances are small planar cationic hydrophobic molecules.
  
  

  - It is energised by ATP hydrolysis. There are two models: flippase (the substrate diffuses laterally in the bilayer); and pump (the substrate binds to a specific site)
  - MDR1 exports lipophilic drugs.
  - MDR2 is involved in the formation of bile in liver, and exports phosphatidylcholine into bile.

Symporters

• Uniporters:- The facilitative diffusion systems. The D-glucose carrier is an antiporter found in virtually all cells, while its mirror image of L-glucose is not transported.
  - Red blood cells are the most studied system, and antiporters have a proteinaceous nature indicated by protein-modifying agents: Protease which also did not affect the membrane barrier; and Cytochalasin B (a product of fungal metabolism helped in selective isolation of the transporter) which is involved in reversible inhibition of the transporter. It also exhibits high affinity binding.
  
  

  Facilitated Transport - Transporters have a specificity for D-hexoses such as 2-deoxyglucose (is phosphorylated and not metabolised further); and 3-O-methyl-D-glucose (is transported but not phosphorylated) which determines the equilibrium exchange and allows bidirectional fluxes to be studied.
  - They have approx 500 amino acids with 12 transmembrane segments.
  - Examples of those expressed on the cell surface include GLUT1 (in erythrocytes); GLUT2 (in liver cells/small intestine/kidney); GLUT3 (in the brain) and GLUT5 (in small intestine).
  - GLUT4 is found in the intracellular vesicles of skeletal muscle
  

Symporters

The glucose transporter structure:
- They are highly homologous with a high sequence similarity. They have 12 amphipathic TM helices with large loops between 1 and 2, and 6 and 7.
- Cytochalasin B binds to the intracellular face between helices 10 and 11.
- Glycosylation occurs at the extracellular loop and is important for membrane targetting to protect against broken down degradation.

• GLUT1:
  - The 'constituitive glucose transporter' is found in erythrocytes (RBCs) and present in many tissues including low levels in the liver.
  - The Km for 3-O-MG (equilibrium exchange) is ~20mM with the unidirectional Km of 1.5mM. The initial rate transport is accelerated when glucose is also present on internal face.
  - It has kinetic asymmetry and allosteric regulation of intracellular glucose speeds up transport, important in glucose starvation.
  - This is upregulated during starvation.

Symporters

• GLUT2:
  - Is found in the liver and pancreatic beta cells and has a Km for 3-O-MG at ~40mM so it is a high capacity high Km (i.e. low affinity) transporter.
  - The unidirectional Km is 20mM, so glucose enters these tissues at a biologically significant rate only when there is much glucose in the blood. The pancreas can thereby sense the glucose level and accordingly adjust the rate of insulin secretion. Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat. The high Km value ensure glucose can rapidly enter liver cells for gluconeogenesis.
  - It also plays a role in kidneys and intestinal epithelial cells where there is uptake of glucose across enterocytes into the bloodstream, and GLUT 2 is found on the apical membrane.
  
• GLUT3:- Is found predominantly in the brain and is responsible for basal glucose uptake with a lower Km of 10mM (so high affinity) and a unidirectional Km for glucose of 1mM.
  - Plays a role in hypoglycemia so that cells can still utilize or catch up low glucose concentrations.
  

Symporters

• GLUT4:
  - Is known as the insulin responsive transporter (upregulated by insulin) which has a unidirectional Km value of 5mM to transport glucose into muscle and adipose tissue. The number of GLUT4 transporters in the plasma membrane increases rapidly in the presence of insulin, which signals the fed state. Hence, insulin promotes the uptake of glucose in muscle and fat cells by 4-fold.
  - There is no change in affinity, but a change in Vmax and thus an increase in the ability to uptake. It ensures a rapid removal of blood glucose to energy stores.
  
  
• GLUT5:- Is present in the small intestine and functions primarily to transport hexoses such as fructose across the apical brush border. It is not sensitive to cytochalasin B.
  
  
• GLUT6 & 7:
  - GLUT6 is not functional, and GLUT7 has found to have an ER role in the liver plus has a high affinity hexose uptake in the small intestine.

Symporters

• GLUT4 Translocation:- In 1980: light microsome fraction vs plasma membrane showed a portion of transporting ability with and without insulin. Photolabeling: An increase in the percentage of transporters was found in the plasma membrane.
  - Re-exocytosis is stimulated, on stimulation by insulin, transporter is inserted in the plasma membrane then it taken back in the cells.
  - Role of clathrin-coated pits (removal of cell surface proteins), chaperone proteins. The rate of exocytosis is greater than endocytosis.
  
  
• Co-Transporters:- This is when a coupled process can harness energy from a transmembrane gradient, i.e. using energy from a sodium ion electrochemical gradient.
  - Na+-Glucose transporters (SGLT) are found on the apical membrane and are used to drive apical glucose entry. This is an electrogenic transporter which generates a current. The rate limiting step is the reorientation of unloaded (negative) carrier. SGLT has two main isoforms, SGLT1 and SGLT2.
  

Symporters

• Na+-Glucose Transporters:
  - SGLT1 is found in enterocytes and has ~14 transmembrane domains. It has a high affinity (Km = ~0.8mM) and is important in the absorption of dietary glucose.
  - SGLT1 is also found in the kidney proximal S3 segment, however in the earlier S1 segment you would find the lower affinity SGLT2 (Km = ~1.5mM).
  - Sodium ions bind to the extracellular face of the transporter causing a conformational change so that glucose can also bind. The conformation changes again to allow the Na+ and glucose to face the intracellular environment. The glucose dissociates first into the lower concentration environment, then the conformation changes again to allow Na+ ions to dissociate.
  - SGLT1 has a high affinity with 2Na+ : 1 Glucose; SGLT2 has a lower affinity with 1Na+ : 1Glu.
  - They act as a key drug development target in diabetes control, e.g. Phlorizin is an inhibitor from plant glycoside and is different from cyt B.
  - Relatives of these include SMIT, a Na+-myoinositol translocator involved in volume regulation; and SNST, a Na+-nucleoside translocator involved in the uptake of adenosine, guanine etc.
  

• Na+-K+-Cl- Cotransporter:
  - Is an electroneutral 12 transmembrane domain carrier which couples Na+, K+ and 2Cl-.
  - It is found in mammalian airways as the basolateral NKCC1, and in the thick ascending limb of the kidney as apical NKCC2.
  - It is regulated by Protein Kinase A which activates the NKCC, and is inhibited by bumetanide.
  
• Na+-HCO3- Cotransporter:- NBC recaptures HCO3- that is filtered in the kidney and transfers it to the blood. It is found in the basolateral membrane of the proximal tubule, and also other tissues.
  - It is electrogenic and Cl- independent, and is also inhibited by stilbenes.
  - The stochiometry can be variable but it is usually 1Na+ to 3HCO3-.
  
  
• Neurotransmitter Cotransporters:- Are plasma membrane transporters which utilize Na+ gradients and also either Cl- or K+.
  Na+/Cl- dependent: - Require Cl- to transport the substrate, all have a high affinity and are electrogenic of 2Na+ to 1Cl-.
  - They transport GABA (via GAT1 which has a Km of 7um so a high affinity - plays a role in epilepsy and anxiety); NA (via NET, with Km = 0.5um, drugs to block transporter are involved in depression); DA (via DAT, Km = 1um, binds cocaine which competes for NET too); 5HT (via SERT, Km = 0.5um, targetted by antidepressants); Glycine (via GLYT, Km = 0.1um).
  

Na+/K+ dependent: - Transports glut- and is electrogenic. It is Cl- independent and works better with a Cl- conductance.
- Recent evidence showing that there is 3Na+ with 1H+ with glut- in exchange for K+.
- It was first cloned as GLAST1 (in rats), GLT-1 (in mice), and EAAC1 (rabbits).
- There are 6 clear membrane spanning alpha helices on the N-terminal. The most common motifs for this group is found in 8 transmembrane domain:
EAAT1 (=GLAST1); EAAT2 (=GLT1) - these two have pathophysiological roles in acute and chronic brain disorders; EAAT3 (=EAAC1); EAAT4 and EAAT5 (astroglial cells).

• Antiporters:
  1) Act as secondary active transport systems, e.g. Na+/H+ exchange and Na+/Ca2+ exchange.
  2) Act to rapidly exchange products of enzyme reactions with their substrates, e.g. Cl-/HCO3-.
  Na+/H+ Exchange: - Electroneutral involved in intracellular pH, cell volume and Na+ transport.
  - There are several isoforms of the NHE family (NHE1-NHE8), and 2 functional clusters with an N-terminal of 500 amino acids, 10-12 TM alpha helices which mediates the exchange. The hydrophilic cytosolic C-terminal consists of 300 amino acids and contains the regulatory domains and volume sensor.
  - NHE1 is the main pH and cell volume regulator.

Na+/H+ Exchange: - NHE2, 3, and 4 are found in kidney epithelial cells and are less sensitive to amiloride (which affects Na+ binding) than NHE1.
- NHE6 is mitochondrial.
- NHE1 is activated by acidosis and volume decrease. Phosphorylation and cell shrinkage increases its affinity to H+. Acidification has an allosteric effect.

Cl-/HCO3- Exchange: - Is electroneutral, AE1 to AE3. Has 13 transmembrane domains.
- AE1 is the smallest and is found in band 3 protein of red blood cells. This plays a role in blood CO2 transport and has a structural role by connecting the membrane to the cytoskeleton.
- AE2 is activated by an increase in intracellular pH and AE3 is found in the brain and excitable tissues of the heart.
- Anhydrase makes HCO3-, H+ binds to haemoglobin and HCO3- is removed in exchange for Cl-. They are inhibited by stilbenes and both NHE1 and AE2 maintain basal pHi.
- They also play a role in volume regulation: Cell shrinkage activates NHE1 which causes alkalinization. This activates AE2. The net result is Na+ and Cl- entry and thus water entry, therefore involved in regulatory volume increase.

Na+/Ca2+ Exchange: - Maintains low Ca2+. Stochiometry is 3 Na+ for 1Ca2+ and so is electrogenic. This functions in the calcium efflux at rest and a calcium influx during action potentials.
- The cardiac isoform has 980 amino acids with 9 transmembrane domains. There is a large loop between M5 and M6 which has a regulatory site.
- The N-terminus is extracellular and there are 3 main members of this: NCX1 found in cardiac tissue involved in long action potentials (plateau phase); NCX2 in the brain and NCX3 in skeletal muscle.
- This plays a key role in cardiac failure and arrythmia.

• CNG and HCN Cation Channels:
  - Cyclic Nucleotide Gated and Hyperpolarization-activated Cyclic Nucleotide-gated channels have shared structural characteristics. They are both composed of 4 subunits (tetrameric) each of which has 6 transmembrane domains. The S5/6 and loop make a pore and S4 contains positive amino acid residues to allow the structure to change its conformation to voltage changes and is thus the voltage sensor. The pore is non-selective for cations (Na+, K+ and Ca2+). Each subunit also has a C-linker and each has an intracellular C and N-terminus, whilst each also has a cyclic nucleotide binding domain.
  - They also, however, have different functional characteristics. CNG channels are activated by cAMP and/or cGMP and thus require these for tonic activity. Whereas, HCN channels are modulated by cAMP and/or cGMP but they do not need these for tonic activity. They are voltage sensitive and open in response to hyperpolarization, but they are less sensitive and also slower than K+ channels.
  - Voltage clamp recordings show that when there is no cNucleotide then there is no response by CNG channels, but there is a response is HCN and a higher current is found at a more negative voltage. In the presence of cGMP, in CNG channels there is an evoked current at each voltage as the voltage increases so does the current. In the presence of cAMP, in HCN channels the size of the current is even larger at each voltage so the voltage dependence has moved so that the channel opens at less hyperpolarised voltages.
  

• Contribution to Physiology:
  Vision - CNG channels are found in the rod photoreceptor membrane and are kept open by cGMP. In the dark there is a tonic depolarization to activate the CNG and activate the rod cell.
  - In the presence of light, there is freezing of the alpha subunit of transducin to inactivate it resulting in the activation of phosphodiesterase which converts cGMP into 5'-GMP causing the channel to close and thus causes hyperpolarization, therefore inactivating the rod cell.
  
  

  Pacemaker - Pacemakers are found in the SAN in the wall of the right atrium. The pacemaker has an unstable resting membrane potential which can reach threshold spontaneously and the rate of depolarization is regulated by the autonomic nervous system. In the sympathetic system the threshold is obtained more rapidly so each cycle takes less time. In the parasympathetic system the pacemaker potential takes longer to reach threshold.
  - Inward rectifying K+ channels set the Vm of excitable cells but SAN cells do not have these, and HCN channels open as repolarization occurs (at approx -50mV) to cause another depolarization of the cell by allowing Na+ ions to enter, when it reaches threshold Ca2+ channels open. cAMP further activates HCN to increase the rate of the threshold reached.
  

• Epithelial Na+ Channels:
  - Contain 4 subunits which each has 2 transmembrane domains. There is usually 2 alpha: 1beta: 1gamma subunits and the alphas can work alone. ENaC is activated by protein kinase A phosphorylation, and each subunit has an intracellular C and N terminus.
  - ENaC is blocked by amiloride and is selective for Na+.
  
• ENaC Contribution to Physiology:
  Birth - Fetal lungs are full of fluid which is actively secreted by the epithelium of the lungs to form a liquid template. Normally term delivered babies have fully reabsorbed fluid, but those that are premature have not.
  - Labour produces adrenaline to the foetus which causes the lung fluid to be rapidly reabsorbed. It binds to B2 receptors at the alveolar epithelium activating cAMP to open the ENaC channel.
  
  
• Transient Receptor Potential Channels:- These are cation selective channels, all of which have 6 transmembrane domains and there are 7 families. They have cytosolic N and C termini and are tetramers found in almost all cells. TRPV1 (vanilloid) contributes to pain as it is a receptor of noxious stimuli. to depolarise the neuron. TRPV1 binds capsaicin to alter the response to heat or acid and therefore this receptor can integrate many stimuli.
  - Capsaicin can also desensitizes TRPV1 so it can be used as an analgesic.
  

In most cells, Cl- is out of equilibrium.

In epithelial cells and immature neurons, Cl- ions are accumulated above equilibrium (which is important for the way in which synapses are formed). There is movement of Cl- ions into the cell via the Na+/Cl- cotransporter and the Na+/Cl-/K+ cotransporter, whilst the Na+/K+ exchange pump is moving K+ into the cell and Na+ out. The Na+ gradient out of the cell drives the two cotransporter to move their ions into the cell. Cl- ions exit the cell via channels but the electrochemical equilibrium is above expected.

In mature neurons, Cl- ions are maintained below the equilibrium. There is entry of Cl- ions into the cell via channels, and the Na+/K+ pump is again producing a Na+ gradient to move Na+ ions into the cell via the Na+/Cl-/HCO3- (NDCBE) exchanger which also moves Cl- ions out. The K+ gradient inside the cell causes movement of K+ ions out of the cell along with Cl- ions with the KCC cotransporter.

• Developmental Regulation of Cl- in Neurons:
  - During development Cl- goes from being accumulated to being removed. In immature neurons the extracellular Na+ gradient formed from the Na+/K+ pump causes Na+ ions to move into the cell via the NKCC cotransporter along with 2Cl- ions, thus there is accumulation. When GABA binds to the GABAa receptor on the membrane it opens to allow the exit of Cl- ions and therefore there is depolarization, thus GABA is excitatory in immature neurons.
  - In mature neurons, GABA activates GABAa receptors to allow Cl- entry into the cell which hyperpolarizes the membrane and thus causes an inhibitory effect. Cl- ions are moved out of the cell via the KCC cotransporter which is activated by the K+ gradient inside the cell caused by the Na+/K+ pump.
  
  
• Cl- Movement Down Electrochemical Gradients:- This is via Cl- channels, including: Ligand-gated (activated by GABA or Glycine extracellularly; CFTR (activated intracellularly by PKA); CICs (activated by H+ or voltage extracellularly, and Cl- or H+ intracellularly); Bestrophins (unknown) and TMEM16/Anoctamins (activate by intracellular Ca2+).

• Cystic Fibrosis:
  - In the normal CFTR, ATP binding drives the channel opening. However, mutations of this receptor (most commonly deltaF508) lead to a loss of function and thus causes CF.
  
  

  - In normal lungs, Cl- ions drive fluid secretion. The Na+/K+ ATPase is causing an extracellular Na+ gradient which moves Na+ ions to cross the membrane into the cell along with 2Cl- via the NKCC cotransporter. There is then an accumulation of Cl- ions above equilibrium so Cl- ions exit the cell via the CFTR into the mucus. This causes Na+ ions to move into the mucus and H2O moves with it to dilute the mucus so that cilia beat efficiently to clear the mucus from the lumen of the airway. The ENaC is then shunted due to Na+ ions passing through the paracellular pathway.

  - In CF lungs, there is still an accumulation of Cl- ions above equilibrium, however the CFTR does not work properly and there is thus less exit of Cl- into the mucus, therefore less water moves into the mucus so there is a reduced airway liquid depth and the cilia will beat poorly to impair the mucus clearance. Mucus then clogs the airways and there is lung failure. ENaC is active so there is hyperabsorption of Na+ ions and there are Na+ ions moving away from the mucus in the lumen of the airway.
  

• CLC-1 in Muscle Physiology:
  - Normal excitation-contraction coupling in skeletal muscle at low activity:
  1) The Na+ pump energizes the cell and moves K+ ions in. The Vm is set at EK by KIR channels through which these K+ ions exit.
  2) Active Cl- transport is low in muscles although there are lots of Cl- channels. Cl- equilibrates passively through CLC-1 channels.
  3) An action potential at the neuromuscular junction activates ACh receptors causing an action potential along the muscle to open voltage-gated Ca2+ channels in the sarcoplasmic reticulum to release calcium ions. These stimulate the release of more calcium ions which result in contraction.
  4) The K+ exit during low activity is small so there is no change in Vm.
  
  

  - At high activity:
  1) There are many muscle action potentials so there is an increased K+ loss to the t tubules and there it can build up and cause more depolarization.
  2) This can be neutralised by the Cl- efflux through a normally functioning ClC1 channel.
  

• Myotonia Congenita:
  - This is a mutation in the CLC1 channel so at high activity there is incorrect gating and an effective loss of function.
  - The increased loss of K+ ions to the t-tubule causes an increased accumulation of K+ ions, but the dysfunctional ClC1 channel does not move Cl- ions into the t tubule to counteract it.
  - Therefore as K+ ions are not neutralised there is tonic depolarization which causes hyperexcitability so the muscles can no longer relax.
  

• Synaptic Transmission:
  There are two main types.
  Electrical - Connexins and pannexins (conducting gap junctions); Protein pores joining two neurons; Fast, reliable transmission; Useful for escape or other rapid responses; De-amplification only; Can promote synchronized firing.
  
  

  Chemical - More versatile information transformation and storage
  - Can flip sign, or have multiple post-synaptic effects; Co-transmission and/or different receptor subtypes/combos.
  - Can introduce time delays or distinct/multiple time-course of effects.
  - Neurotransmitter release is calcium dependent but also a baseline calcium-independent component.
  - Synaptic vesicles have a probabilistic 'quantal' release.
  - Amplification/attenuation step (encodes saliency)
  - Unreliable transmission (encodes probability)
  - Important source of noise, vital for neural computations.
  - Some chemical synapses are reliable, large with lots of vesicles.
  

• Electrical Synapses:
  - There is a gap junction between the presynaptic and postsynaptic neurons and ions flow through the channels to the postsynapse.
  - There are pores connecting the cytoplasms of the two neurons consisting of  6 connexons. The pore allows conductance. V1 is the membrane potential of the first cell, and V2 is the membrane potential of the second cell. I = g(V1 - V2).
  - Gap junctions can promote synchrony. Metabolic glutamate agonist causing oscillations. Cross-correlograms are a standard tool for quantifying the degree of synchrony.
  
  
• Chemical Synapses:
  1) Transmitter is synthesised and then stored in vesicles.
  2) An action potential invades the presynaptic terminal.
  3) Depolarisation of presynaptic terminal causes opening of voltage-gated calcium channels.
  4) Influx of calcium ions through channels causing vesicles to fuse with the presynapse.
  6) Transmitter is released into the synaptic cleft via exocytosis.
  7) Transmitter binds to receptor molecules on the postsynaptic membrane.
  8) Opening or closing of postsynaptic channels. Postsynaptic current causes excitatory or inhibitory postsynaptic potential that changes the excitability of the postsynaptic cell.
  9) Retrieval of vesicular membrane from plasma membrane.

• Chemical Synapses:
  Vesicle Cycle - Vesicles are formed from endosomes and held together with synapsin, then they are docked onto the presynaptic membrane.
  - Using NSF and SNAPs the vesicles are primed so that SNAREs hold them onto the membrane.
  - Calcium ions enter the cell to fuse the vesicles with the membrane via calcium binding to synaptotagmin (the calcium ion sensor).
  - After exocytosis the vesicle proteins remain clustered and are then retrieved by endocytosis. Retrieval is mediated by clathrin-mediated endocytosis.
  - After clathrin uncoating, synaptic vesicles are regenerated within the nerve terminal, probably involving passage through an endosomal intermediate.
  - Actively recycling vesicles are in slow exchange with the reserve pool.
  
  

  Neurotransmitter Recycling - Glutamate is taken up into glial cells, converted to glutamine, which then recycles back to the neuron via transporter proteins.
  - Glutamate has a negative charge, so the positive charge gradient across the vesicle membrane from outside to inside can power the VGLUT vesicular glutamate transporter, and allows it to build up very high concentrations of glutamate in the vesicle.
  

There are 3+ different pools of vesicles: readily-releasable; reserve (not in active zone) and recycling.

3+ different types of vesicle release: 1. Calcium-dependent synchronous release which uses SNARE protein VAMP2; 2. Asynchronous release (increases with residual calcium ions but not tied to AP times, relies on VAMP4 protein as alternative to calcium ion sensor; 3. Spontaneous.- Full release (collapse of vesicle, slow recycling); Kiss and run (transient opening of fusion pore, incomplete emptying).

• Quantal Release : At a single synapse, post synaptic electrical respones to a single pre-synaptic action potential appears to jump between integer multiples of a discrete size. Often see total release failures.
• Miniature Postsynaptic potentials:Occur in background without presynaptic action potetial, due to spontaneous vesicle fusion.
  - Gap between peaks = average quantal size, or postsynaptic voltage resulting from single vesicle release.
  

• Pathologies of Release:
  Tetanus toxin - A-chain attacks the vesicle associated membrane protein (VAMP) SNARE. Stops affected neurons from releasing the inhibitory neurotransmitters GABA and glycine by degrading the protein synaptobrevin.
  - Causing dangerous overactivity in muscles, failure of inhibition of motor reflexes by sensory stimulation causing generalized contractions of the agonist and antagonist musculature.
  Clostridial toxins - A bacterial protease cleaving SNARE proteins. botulinum neurotoxin (botox).
  - These attack release machinery, to block the release of ACh and other transmitters.
  
  
• 'Atypical' transmitter- ATP coreleased from synaptic vesicles, broken down to adenosine which acts on presynaptic adenosine receptors.