Voltage-gated Ion Channels Flashcards
6TM1P
six transmembrane, 1 pore loop (between S5 and S6)
-alpha-1 subunits are comprised of 4 of these domains
S4 transmembrane domain
polarized alpha helix, voltage sensor, part of activation gating mechanism
P-loop domain (S5, S6, and P-loop)
pore forming, ion selectivity
S1-3 transmembrane domains
non-polarized alpha helices, “hydrophobic mantle” for S4 and P-loop domains in gating process
what influences the state transitions of voltage-gated ion channels?
time and voltage
opamp 1
detects voltage difference between Vm and ground
opamp 2
compare voltage input from opamp 1 with voltage set by experimenter (Vh)
the principle of voltage clamp
1) cell generates currents (Im) causing change in Vm
2) opamp 1 measures Vm
3) output from opamp 1 is sent to opamp 2’s “inverting” input
4) opamp 2 compares voltage input from opamp 1 with voltage set by experimenter
5) current generated by cell (Im) is neutralized by current generated by opamp 2 (Ic)
two-electrode voltage clamp
separate voltage-sensing and current injecting electrodes
patch clamp (single electrode voltage clamp)
low resistance electrode with high impedance feedback resistor
patch clamp depends on formation of:
high resistance cell - “gigaseal”
cell-attached recording
cell-attached recording (patch clamp)
suction to form a gigaseal with the membrane (high resistance)
whole cell recordings (patch clamp)
- suction to form a gigaseal
- if using a small cell, pulse of suction ruptures the membrane patch
outside-out patch (patch clamp)
- suction to form a gigaseal
- pulse of suction ruptures the membrane patch
- pull electrode out until cytoplasmic bridge collapses
- outside leaflet of membrane faces outwards from the electrode
inside-out patch (patch clamp)
- suction to form a gigaseal
- pull electrode until until cytoplasmic bridge collapses
- pull in low calcium medium or air exposure breaks out the cell patch
- outside leaflet of cell membrane faces inwards towards the electrode
open probability
time channel is open divided by total observation time
“microstates”
single channel currents
“macrostates”
whole cell currents (the sum of individual channel currents)
what are 3 strategies for separating total cell current into independent components?
1) ion replacement strategies
2) pharmacological blocking of ion channels
3) conditioning prepulse protocols
alpha-conotoxin
voltage-gated calcium channel blockers
u-conotoxin
acetylcholine receptor blockers
w-conotoxin
voltage-gated calcium channel blockers
delta-conotoxin
voltage-gated sodium channel blockers
k-conotoxin
voltage-gated potassium channel blockers
what subunit is responsible for inactivation of some voltage-gated potassium channels?
the N-terminal of some Kv-a subunits
how to find IA (current from activation of a voltage-gated potassium channel) using conditioning prepulses?
1) isolate delayed-rectifier current (IDR) after inactivation following a depolarized conditioning pulse
2) find IK (total K+ current) using a voltage-clamp protocol
3) IA=IK - IDR
what determines the shape of an I/V curve?
the effect of membrane potential on ion channel opening (G-conductance of channel) and driving force of the permeant ion (Vm-E Nernst)
tail currents
measures the effect of voltage-dependency on channel gating without the effects of an electrochemical driving force, measures instantaneous or tail currents at a fixed voltage so that driving force is constant
- variable holding potential followed by a return to a fixed voltage
- can be used to plot instantaneous current (current when switched to holding potential is proportional to the number of channels open immediately before the voltage step) against the voltage during the first depolarization
how do you describe/analyze voltage-dependencies of whole cell currents?
statistical mechanics allows you to predict and explain the measurable properties of macroscopic systems on the basis of the properties and behaviour of the microscopic constituents of those systems
Maxwell-Boltzmann distribution
links probabilistic behaviour of individual gas molecules to the global observable behaviour
Boltzmann equation
the bridge between ion channel dynamics and whole cell current
Vh (Boltzmann equation)
half-maximal voltage: the membrane voltage at which half activation (or inactivation) occurs
k (Boltzmann equation)
steepness factor: determines how steeply the global (in)activation curve changes with voltage, proportional to equivalent number of charges on the channel protein gating particle (S4)
activation curve
instantaneous current (current when switched to holding potential is proportional to the number of channels open immediately before the voltage step) is plotted against voltage during the first depolarization
inactivation curve
the current flowing during the test pulse is expressed relative to its size in the absence of a prepulse (I/Imax) and plotted against the prepulse potential
how can you investigate the time course of the development of inactivation at a particular potential?
two-pulse protocol: use a prepulse of fixed voltage but whose duration is varied
how can you investigate recovery from inactivation?
two-pulse protocol: vary the duration of the interval between a pre-pulse and a test-pulse
window current
refers to a property of some intrinsically inactivating voltage-gated ion channels to allow persistent current flow over a small intermediate range of their full voltage operating range
what are the 2 main classes of voltage-gated calcium channels?
1) high-voltage activated calcium channels (HVA)
2) low-voltage activated calcium channels (LVA)
L-type calcium channels
HVA; CaV1.1-1.4; moderate/slow inactivation; calcium-dependent inactivation present
P/Q calcium channels
HVA; CaV2.1; moderate/slow inactivation; calcium-dependent inactivation may be present
N-type calcium channels
HVA; CaV2.2; fast inactivation; calcium-dependent inactivation may be present
R-type calcium channels
HVA; CaV2.3; fast/moderate inactivation; no calcium-dependent inactivation
T-type calcium channels
LVA; Ca3.1-3.3; very fast inactivation; no calcium-dependent inactivation
nifedipine
L-type calcium channel blocker
heavy metals (NiCl2, CdCl2)
blocks all types of voltage-gated calcium channels
w-CgTX (conotoxin) GVIA or MVIIC
N-type calcium channel blocker
what are the 2 different forms of inactivation exhibited by voltage-gated calcium channels?
1) voltage-dependent inactivation (VDI) - change in membrane potential causes channel inactivation
2) calcium-dependent inactivation (CDI) - increase in intracellular calcium causes channel inactivation
