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
Equilibrium potentials for each ion
Dependent on ionic distribution, each cell type has its own equilibrium potential for each ion species.
Nernst equation determines the equilibrium potential for membrane permeable only to one type of ion
