AMPA receptors
permeable to Na+ (+55mV) and K+ (-90mV)
- slightly more permeable to Na+ than K+
- transient current
leak K+ current
persistent K+ current, moderate permeability
leak Na+ current
persistent Na+ current, low permeability
tonic GABA current
persistent Cl- current , low permeability
voltage-gated Na+ current
transient Na+ current, high permeability
low intracellular chloride concentration results in:
hyperpolarized chloride Nernst potential (more outward current)
high intracellular chloride concentration results in:
depolarized chloride Nernst potential (more inward currents)
potassium chloride cotransporter 2 (KCC2)
maintains intracellular chloride levels (symport of chloride and potassium ions out of cell)
-KCC2-mediated chloride efflux driving force increases as intracellular chloride concentration increases
what are 2 requirements for measuring KCC2 activity?
1) a method to elevate intracellular chloride levels (chloride load = increase KCC2 activity)
2) a method to track chloride gradients
halorhodopsin
inward chloride pump that is activated by yellow light (595nm), can elevate chloride levels to measure KCC2 activity
what is a method to track chloride gradients?
voltage clamp (halorhodopsin photocurrents transiently reduce the amplitude of GABA puff-induced currents/chloride influx) -more positive E gaba, amplitude of current decreases
what is the goal of electrophysiology?
to measure and manipulate the electrical properties of cells (voltage and current)
voltage
the difference in charge between two compartments (with respect to the extracellular space)
negative voltage
if inside of cell is more negative than outside of cell
positive voltage
if inside of cell is more positive than outside of cell
current
the movement of charge between two compartments
inward current
the introduction of positive charge into the cell (cations move in, anions move out) - leads to depolarization
outward current
the introduction of negative charge into the cell (cations move out, anions move in) - leads to hyperpolarization
whole-cell electrophysiology
measure voltage between the extracellular and intracellular space, inject positive or negative charge, measure amount of injected current
current clamp
study alterations of the membrane potential during neurological processes: injected current (we choose how much current we inject into the cell) and measure voltage
voltage clamp
study the ionic currents generated by ion channels/pumps/transporters: clamp voltage (choose membrane potential that we want the cell to stay at), measure the amount of injected current required to maintain the voltage
inject positive current
depolarization of membrane potential
inject negative current
hyperpolarization of membrane potential
potassium leak channels
outward current at -30mV, persistent
GABA-A channels
outward current at -30mV, transient
capacitive current
spike when clamp potential changes, direction of current same as direction of voltage change
voltage-sensing channel
measures the voltage between the voltage electrode and the bath electrode
electrode test (voltage-sensing channel)
inject 10nA of current, measure voltage drop (use V=IR to determine electrode resistance)
BUZZ pushbuttons (voltage-sensing channel)
facilitates penetration of the cell membrane
negative capacitance compensation (voltage-sensing channel)
allows you to discount the current charging the glass pipette (chloride/current entering the voltage electrode can go to the AgCl wire or to the glass pipette)
current-sensing channel
depending on the settings, can 1) measure the current injected by the current electrode during voltage clamp 2) measure voltage
clamp speed
refers to the time it takes for the amplifier to clamp the voltage (limited by how frequently the membrane potential is measured)
clamp selector: off
the current electrode measures voltage
clamp selector: slow
the current electrode measures current (clamp speed is 0.5ms)
clamp selector: fast
the current electrode measures current (clamp speed is 350 us)
gain
amplifies the data: every data point is multiplied by the gain (amplifies both the signal and noise)
current clamp channel
delivers current into the cell
over voltage (current-clamp channel)
alarm sounds if the voltage at the current electrode exceeds +/- 160V
gain telegraph output BNCs (rear panel)
outputs the gain of the amplifier
alarm switch (rear panel)
activates or inactivates the audible alarm for the over voltage warning
grounds (rear panel)
grounds the electronic
the voltage probe
connects to the voltage electrode control box, the electrode of the voltage probe must be coated with bleach (chlorine)
current electrode cable
connects to the current electrode control box, the electrode of the current electrode cable must be coated with bleach (chlorine)
