Study of Ionic Currents by the Patch Clamp

Study of Ionic Currents by the Patch Clamp Technique

The patch clamp method • Certain membrane region is electrically separated from neighbouring regions by gigaseal (R >109 Ω) • There are several ways to get a membrane patch • Often voltage clamp and patch clamp are combined into one method • Glass pipettes serve as electrodes for patch clamp and by the gigaseal the distance between pepette tip and membrane < 1 nm • The patch clamp method gives a possibility to measure currents going through a single or few ion channels • As ion channels are in the membrane of every cell, the patch clamp technique is usable to study of any cell

The patch clamp circuit The amplifier compares the membrane potential (Vm) to the new command potential (Vcmd) specified by the operator as -20 m. V

The patch clamp circuit The difference between Vm and Vcmd is corrected by injecting Vo down the micropipette. This depolarizes the membrane and voltage gated channels open. The current flowing through a single channel is Ip

The patch clamp circuit The current passing through all the channels (Ip) flows through the circuit and is measured as a voltage change. Vo= -Rf· Ip + Vcmd -Ip · Rf = Vo- Vcmd Rf (feedback resistor) determines the sensititvity, range of current measurement, and the background noise level. Usually Rf is 5 -10 GΩ

The Setup of Sim. Patch • Patch-clamp amplifier • Stimulator or pulse generator • Oscilloscope • Cell(s), electrodes and headstage amplifier are missing on the screen • The bottom line buttons are for management of and to use additional facilities of the virtual system

Patch clamp • Macrocellular currents – Whole cell • Microcellular currents – Inside-out – Outside-out

Patch clamp • Patch clamp allows measurements of electrical currents and potentials from the entire cell. • Also allows diffusion between cell and pipette so that drugs can be injected into the cell.

Patch clamp • Inside out recording allows measurement of single channel currents and allows to change the chemicals exposed to the intracellular surface. Useful in studying second messenger actions on channels. • Outside-out allows to study the effects of extracellular chemicals as occurs in cellular signaling.

Patch clamp technique

Currents The currents flowing through single channels are called microscopic current to distinguish from macroscopic currents flowing through a large number of channels distributed over a much more extensive region of surface membrane

Microscopic and macroscopic currents: Na+

Regenerative Process: Once one Na channel Opens, Na enters, Depolarizes membrane, More and more Na Channels open leading to More sodium influx & causes upward & depolarizing (more +) phase of the AP

Property of Voltage Dependent Sodium Channel • Sodium channel opens for 1 -2 millisecond following threshold depolarization. • Then inactivates and does not open even if Vm is depolarized. • This is called sodium channel inactivation and contributes to the repolarization of Vm.

Na Channel Gates • M gate= activation gate on Na channel; opens quickly when membrane is depolarized • H gate- inactivation gate on Na channel; Closes slowly after membrane is depolarizedcauses the absolute refractory period for AP propagation

Microscopic and macroscopic currents: K+

Potassium Channel Property • K channels open with a delay and stay open for length of depolarization. • Repolarize the Vm to Ek= -75 m. V which is why you have hyperpolarization. • Also called a delayed rectifier channel.

Gate on the Delayed Rectifier Potassium Channel • K channels have a single gate (n) that stays open as long as Vm is depolarized. • n gate on K channels opens very slowly this allows the Vm to depolarize due to Na influx; Na and K currents do not offset each other right away.

Voltage-gated channels

Voltage-gated channels • Up to 10 different Na channels in mammals. Some Na channels are rapidly inactivating and some are not. Those that do not rapidly inactivate produce APs of longer duration. This channel is a target of local anesthetics benzocaine and lidocaines. • Ca channels can lead to production of APs or shape an AP. It is also involved intracellular signalling. 16 different Ca channel genes have been identified. • There are nearly 100 K channels that differ in activation, gating, and inactivation. Set resting membrane potential—two K+ channels are open at negative voltages. • Cl- channels are present in every type of neuron. They control excitability, contribute to resting Vm and regulate cell volume.

what happens to the form Of the action potential When you add a voltage Sensitive calcium channl

Ligand-gated channels

Chemical Synapse 1) Arrival of an action potential at the synaptic terminal triggers opening of voltage gated Ca 2+ channels and the influx of Ca 2+ ions into the terminal. Elevated Ca 2+ in the synaptic terminal causes synaptic vesicles to fuse with the presynaptic membrane. 2) Vesicles then dump their contents into the synaptic cleft.

Chemically Gated Ion Channels 3) Neurotransmitters then diffuse across the synaptic cleft and bind with receptors on chemically gated ion channels in the postsynaptic membrane. 4) Binding of neurotransmitters to the receptors opens the ion channel allowing ions (here Na+) to diffuse into the postsynaptic cell, depolarizing it in this example.

Chemically Gated Ion Channels 5) The neurotransmitter is either degraded by an enzyme or is taken up by the presynaptic neuron causing bound transmitters to dissociate from the ion channels. The channel closes when the neurotransmitter is no longer bound and the postsynaptic cell begins to return to its resting condition.

Other channels • Heat activated ion channels • Temperature activated ion channels • Activation of channels through membrane distortion • Auditory hair cells have directional properties that open and close channels • Light activated channels • Olfactory gated channels

What does a sodium Channel look like? It is one large protein With 4 domains that Each loop through the Plasma membrane 7 Times.

• Both Na and Ca channels have 6 membrane spanning regions that are repeated 4 times. Typically two membrane spanning domains form the pore through which ions travel and one domain contains a protein loop that confers ion selectivity. • The channel binding sites can bind drugs or toxins. • VG channels generally have positively charged amino acids along one face ( in yellow).

Voltage sensing channels

The voltage sensor pulls the S 4 -S 5 linker, which opens the channel pore. Hyperpolarization pulls the linker down.

Active tranporters • Maintain the chemical and electrical gradient • Movement of ions is slower than through channels • Gradients produced by active transporters store energy – Open channels dissipate energy • Two types of transporters—ATPase and ion exchanges

ATPase transporters • Uses 20 -40% of energy in brain • More Na+ is transported than K+ • Has small effects on Vm • Has a larger affect on small diameter axons

• Discovered in the 50 s (Richard Keynes) by looking at radioactive Na efflux from the squid giant axon. Keynes found that the efflux stopped when ATP production was stopped by metabolic poisons. Studies radioactive K+ showed that Na efflux is associated with K influx and that the two are inseparable. • Because less K is pumped out than Na the pump is said to be electrogenic, it produces a small current and affects resting potential by only a m. V or less.

Blocking the pump Prolonged stimulation of small axons inhibits the pump and prevents hyperpolarization. Blocking the pump with ouibain prevents hyperpolarization.

Molecular organization of pump Has two subunits alpha and beta. Alpha spans the membrane 10 times and beta spans it once Sites that move Na and K are not identified but it is thought the ions bind to the same site and that they are negatively charged. Removing the negative charge reduces the effectiveness of the pump.