ELM12: Electrophysiology Flashcards
What is the overall idea of electrophysiology?
Measuring the current between a sample and surrounding medium to find flow and charge
What are some advantages of electrophysiology?
Measure events on timescale
Measure activity of single ion channel
Good spatial resolution
Tells how channels work
What are the three types of electrophysiology?
Extracellular
Intracellular
Patch
What is extracellular electrophysiology?
Electrode outside next to cell
Easy
Non specific
EEG
What is intracellular electrophysiology?
Through membrane
Hard
Specific
What is patch electrophysiology?
Very hard and specific
Record many and single channels
How does patch electrophysiology work?
- A glass tube is put in heating element
- Ends of tube pulled which pulls tube apart into small point
- Glass spike painted with resin and heated to set
- End heated to smooth glass
- Patch clamp rig puts in right place
What are the four types of patch clamping?
Cell attached
Whole cell
Inside out
Outside out
What is cell attached recording?
Electrode put into surface and suction
Trap channels to record only one
What are the applications of cell attached recording?
Record currents through active channels
Single channel currents response to regulation of channels
What is whole cell recording?
Do cell attached and suction more to disrupt membrane
Inside electrode continuous to inside cell
What are the applications of whole cell recording?
Drug screening
Record currents through active channels in whole cell
What is an inside out recording?
- Cell attached mode in bath
- Pull membrane away so a piece is inside electrode
- Pull out of bath so ends of membrane seal to electrodes
- Put back in bath
What are the applications of inside out recording?
Record currents through single channel away from cell
Look at agents that modulate channel at intracellular face
What is an outside out recording?
- Do whole cell recording where membrane breaks
- Rip broken piece off and keep in bath
- Two ends seal to each other
What are the applications of outside out recordings?
Record currents through single channel away from cell
Agents that modulate channel at external face
What does the k-1 rate constant determine?
Stability of open state
Big value means brief openings
What does the k+1 value determine?
Stability of closed state
Big value causes brief closings
What determines the size of the current when ions move across membrane?
Number of ions per second that cross
Ion concentration
Voltage
Pore properties
What is condutance?
The ability of a channel to move ions through the membrane
What does high conductance result in?
Large current blip
What does low conductance result in?
Small current blip
What was the process of an experiment into determining conductance?
- Inside out patch used to look at potassium channel
- Ion concentration on either side is different
- Different recordings of different membrane potentials
What does the gradient of the line tell us on a recording of membrane potentials?
Information about how membrane potential affects the current
What is the method used to measure voltage and current in a large cell?
Two electrode voltage clamp
How does the two electrode voltage clamp work?
- Two electrodes inserted into cell
- Voltage electrode measures membrane potential
- Current electrode injects current to maintain clamp
- Oocytes put in small chamber with buffer and drugs applied
How is patch clamping used on brain slices?
- Apply positive pressure to fine glass recording electrode
- Jet of solution comes out electrode tip and moves debris away
- Use microscope or see charge to know when cell is under electrode tip
- Suction applied and recordings made
How is a patch clamp technique combined with RT PCR?
- Contents of electrode after patch clamp transferred to test tube
- Conversion of mRNAs to cDNAs using reverse transcriptase
- Add primers and cDNA amplified and measured
