Patch clamp electrophysiology and single channel recording Flashcards
Electrophysiology definition and the Nernst equation
Electrophysiology is the measurement of moving charge. A difference in the concentrations of different ionic species on either side of the lipid bilayer causes a potential difference.
The equilibrium potential of a charged species (Q) is defined by the Nernst equation. Alternatively the Nernst equation can be considered as the reversal potential: the point when an ion will shift from moving in/out of the cell.
Resistors and capacitors and their metaphoric equivalents in cells
Any conductor that limits current flow through it is a resistor. Alternatively, conductance can be considered the amount of current flow possible through a conductor. this is reciprocal to resistance. Ion channels can be considered as resistors across the membrane. They have defined conductance associated with each individual ion channel. Ohms is resistance unit. Siemens are unit for conductance. Reciprocal of resistance
A capacitor is a device capable of storing charge. upon change to the potential, the charge can flow out of it. Farads unit.
Lipid bilayer acts as a capacitor. It is dielectric - an electrical field is maintained around the membrane. Upon ion channels opening, ions from the capacitor flow through, but the capacitor quickly reaches a new equilibrium.
Current and voltage clamp
In both a micro electrode is inserted into the cell.
In current clamp, changes to the cell membrane potential over time are measured by the microelectrode.
In voltage clamp, the electrode is fixed to a specific voltage. This maintains the cells membrane potential by injecting current into the cell. The amount of current injected into the cell is the opposite of the amount of current that flows into the cell through ion channels.
Cons of micro electrode recordings and fluctuation analysis
Can only measure the sum of all the ion flow within the cell. All the different ion channels and species.
As the electrode pierces the cell, it harms the cell and can lead to leaks through the hole.
Cannot identify the number of channels, and their respective conductance, open time, and probability of opening.
Statistical analysis (fluctuation “noise” analysis) compares the variance to the mean current.
Patch clamp technique and configurations
Uses a polished glass micropipette, which forms a suction seal against the cell surface. No piercing. Can record single channels. Can measure single gating events.
Cell attached described above. Forms gigaseal
Inside-out patch is were a segment of the membrane is pulled out from the gigaseal.
Whole-cell is done by applying a strong suction to the gigaseal, which ruptures the cell membrane. Can measure the whole cell interior.
Outside-out is done by pulling the pipette from the whole cell configuration, which leads to the membrane to come off. The two segments of membrane will come together and reform. It will be in the opposite orientation to the inside-out.
The power of single channel recordings
Can measure individual molecules operating in real time.
Measures unitary channel conductance.
Measures probability of ion channel opening.
Measures the kinetics of the ion channel: Gating, open/close speeds, sub-conductance, and desensitisation
State transitions and characteristics of ion flow through open states
The transitions between states is very rapid. The conformational change from open to closed or vice versa is faster than the resolution of the microelectrode recordings.
Ion flow through the channel happens in bursts, which generally cluster together.
Stochastic nature of ion channels opening
Individual ion channels appear to open and close at random. Proved by exponential probability distributions of ion channel opening.
When large numbers of the channels are summed together however, they result in organised and predictable synaptic transients.
