Objective 2: Describe the methodology and data arising from current and voltage clamp electrophysiology AND describe properties of voltage-gated ion channels.

METHODOLOGY IN ELECTOPHYSIOLOGY

Current clamp: inject a known quantity of current into the cell, measure the Vm response.
Voltage clamp: used to investigate the currents flowing through individual ion channels. Here, the voltage across the cell membrane is monitored while simultaneously injecting a small amount of current to clamp the transmembrane voltage at a desired level.
- Analysis of this type has shown that Na+ channel activation gates have fast kinetics, are closed at resting potential and open in response to depolarization. Also, K+ channel activation gates are the same but have slow kinetics.
Patch clamp: This involves placing the electrode tip on the membrane of the cell to form a high resistance seal between the wals of the tip and the membrane. This allows one to measure the activity of single channels incorportated into the membrane trapped on the tip of the electrode.
Whole cell patch clamp: A pulse of suction applied to a patch clamp ruptures the membrane and allows whole cell recording. Properties of one type of channel can be studied by blocking other types with toxins/drugs.

VOLTAGE-GATED ION CHANNELS: Na+

General structure:

Consist of large alpha subunits that associate with proteins. The subunit has 4 repeat domains I through IV, each containing 6 membrane-spanning segments S1 through S6.
The S4 segment is highly conserved and acts as the channel's voltage sensor. The sensitivity of this channel is due to positive amino acids located at every third position. When stimulated by a change in transmembrane voltage, this segment moves toward the extra cellular side, allowing the channel to become more permeable to ions.
There are 3 main conformational states: closed, open and inactivated.
Before an action potential occurs, the channels are in their deactivated state, blocked on the extracellular side by their activation gates.
When an action potential occurs, the activation gates open and Na+ ions flow into the neuron, causing depolarization of the membrane.
At the peak of an action potential, the channels inactivate themselves by closing their inactivation gates, which can be thought of as a plug tethered to domains III and IV.

Data from experiments:

Activation gate: fast kinetics, closed at resting, opens in response to depolarization.
Inactivation gate: slow kinetics, open at resting potential, closes in response to depolarization.

VOLTAGE GATED ION CHANNELS: K+

ACTION POTENTIAL MECHANISM

Hodgkin and Huxley used the giant squid axon to make early recordings of action potential and show the role of Na+ and K+.
The action potential is choreographed by the activation threshold, ion selectivity and inactivation of Na+ and K+ voltage-dependent ion channels.
- molecular mechanisms of an action potential: https://www.coursera.org/learn/medical-neuroscience/lecture/HLwlx/molecular-mechanisms-of-action-potential-generation-part-1

	Criado por Georgia Murphy mais de 6 anos atrás

Notes on lectures 1-4 covered by Gareth Evans