W6L1 Flashcards
Patch clamp - Two types of recording
- Current-clamp (I-clamp)
- record voltage (commonly in mV)
- close to physiological conditions - Voltage-clamp (V-clamp)
- record current (commonly in pA, nA)
- Voltage is under control
I = GV
I-clamp record
Can inject current
- causes membrane depolarization
Measure voltage
V-clamp record
Can inject voltage
Measure current
For…
- A-type K+ channel, current goes upwards. Reaches a peak and gradually comes down, due to the ball and chain model of the A-type K+ channel
- Na+ channel, current goes down (inward current), then reach peak very quickly, then becomes inactivated, then current gradually increases back up.
- channel is monomer
4 configurations of patch recordings
- Cell-attached patch recording
- only focus on the channel underneath your patch
- looks like the pimple suction tool - Whole cell recording
- looks like the pimple suction tool but the pimple ruptured - Excised inside-out patch
- when you pull the patch from ‘Cell-attached patch recording’ away from the cell
- inside of the cell faces the bath solution now - Excised outside-out patch
- extracellular side of the membrane faces outward (not facing towards the patch recorder)
Advantages and disadvantages - patch clamp
Advantages
- Good for V-clamp, quantitative.
- able to measure single channel activities in some configurations.
Limitations
- washout of cell content in certain configurations
– could mix with pipette solution or other stuff
– occurs for all 4 configurations except Cell-attached patch recording
A single voltage-gated Na+ channel
- input membrane potential in to depolarize membrane a bit
- measure patch current (pA)
- Aggregate current
- only has current near beginning of voltage input, gradually decreases as time goes on
- shows that channel is mostly open at the beginning
Patch clamp recording of single channels
Staircase
- one channel open vs two channels open
Single channel current is constant (at fixed voltage)
Random appearance of channel openings and closings
The above current transitions are essentially instantaneous
- When held at membrane potential of 0 mV, unitary currents are ~10 pA (i.e. 10 x 10-12 amperes)
- This corresponds roughly to 100,000,000 K+ ions per second traveling through the channel
Summary of conventions you will encounter
Separation of charges gives rise to a potential, or voltage gradient
Similar charges repel one another, opposite charges are attracted.
Permeable and semi-permeable membranes.
Diffusion coefficient, D, a parameter to describe how fast an ion (or atom) can defuse through a membrane barrier.
Forces on an ion
- Chemical force
- Chemical force depends on the concentration gradient and absolute temperature (T) - Electrical force
- Electrical force depends on the charge and electrical gradient (potential)
Total force = electrical force + chemical force
electrochemical equilibrium
electrical force = chemical force
Equilibrium potential of K+ is described by the Nernst equation
Ek = (RT/zF)*ln ([K]2/[K]1)
units are V
Ek = equilibrium potential for K+
R = universal gas constant (2 calories/degree K x mole)
T = absolute temperature (degrees Kelvin: 273 + degrees celsius)
z = valence of ion (K+ = +1)
F = the Faraday’s contant (# charges per mole: 96487 C/mole, equivalent to 2.3*10^4 calories/V x mole)
[K]2 = concentration of K+ in compartment 2
[K]1 = concentration of K+ in compartment 1
Calculating Ek+
At 27oC, T = 300 Kelvin
z for K+ = +1
ln X = ln10*log X
Ek = 0.06 log [K]2/[K]1
Units are V
Ek = 60 log [K]2/[K]1
Units are mV
Nernst equation for ion X
Ek = (RT/zF)*ln ([K]o/[K]i)
EX = equilibrium potential for X ion
z = charge of diffusible ion (e.g. K+ = +1, Cl- = -1, Ca2+ = +2)
R = universal gas constant (2 calories/degrees K x mole)
T = temperature (absolute! degrees Kelvin. That is, 273 + degrees C)
F = Faraday’s Constant (# charges per mole: 96,487 C/mole which can
also be expressed as 2.3 x 104 calories/V x mole)
Ion distribution in mammalian muscle
Note: concentrations are in mM. When you consider Ca2+, the cytosolic free concentration is 100 nM, or 1 x 10-7 Molar, or 10-4 mM)
- Na+
- extracellular: 140
- intracellular: 14 - K+
- extracellular: 5
- intracellular: 150 - Cl-
- extracellular: 135
- intracellular: 10 to 30 - Mg++
- extracellular: 1
- intracellular: 0.5 - Ca++
- extracellular: 2.5
- intracellular: 0.0001 - H+
- extracellular: 4.5-5 (pH 7.4)
- intracellular:710^-5 (ph 7.2) - Osmolarity (mOsm/L)
- extracellular: 295
- intracellular: 295
Equilibrium potentials for each ion
Dependent on ionic distribution, each cell type has its own equilibrium potential for each ion species. Common values are:
EK+ -89 ~ -100 mV
ENa+ +45 ~ +60 mV
ECl- -65 ~ -75 mV
