2. Lectures 3, 4, 5

Question 1

What is the membrane potential?

Answer 1

Result of a separation of charges across cell membrane (works as barrier and capacitor - ability to separate charges)
Excess positive outside membrane (high Na+ and Cl-)
Excess negative inside membrane (K+ and inorganic ions (A-) aminos and proteins
Membrane potential depends primarily on its permeability to Na and K

Slide 4 Lectures 3/4

Question 2

How do we record the membrane potential?

What is hyperpolarization and depolarization?

Answer 2

Vm=Vin-Vout
Vm= membrane potential
Vin= potential in the inside of cell
Vout= potential on outside of cell (experimentally considered zero)

Record charges at membrane

Depolarization- anything from resting potential (-60mV) and up (positive)
Hyperpolarization- anything from -60mV and down (negative)

Slides 5-7 lectures 3/4

Question 3

What is the Nernst equation?

Answer 3

Ex=58mV/z log10([X]o/[X]i)
Ex= equilibrium potential for ion X
z= valence of the ion
[X]o= extracellular conc of ion
[X]i= intracellular conc of ion

All we need to know is valence and internal and external concentrations

Slides 9-15 Lectures 3/4

Slide 39 Lectures 3/4 Q1

Question 4

How is the electrochemical gradient of Na+, K+, and Ca2+ maintained?
Why is this key?

Answer 4

Active transport of the ions creates the electrochemical gradient

Separation of charged must be maintained constant, pumps prevent dissipation of ionic gradients

Slide 16 lectures 3/4

Question 5

How does Cl- (chloride) contribute to the resting membrane potential?
(Nerve cells vs neurons)
What kind of transporters?

Answer 5

In most nerve cells Cl- gradient is controlled by one or more active transport mechanisms
In neutrons it is determined by cotransporters that move Cl- out of the cell

K+ - Cl- cotransporter- Cl moves out, resulting in high [Cl-] out (activation of Cl channels leads to hyperpolarization consequence)
Na+ - K+ - Cl- cotransporter- Cl moves in, resulting in high [Cl] in (activation of Cl channels leads to depolarization consequence)

Early neuronal development shows only NaKCl cotransporters then as they develop they begin to express KCl cotransporter

Slide 17 Lectures 3/4

Question 6

What pumps performance can trigger seizures?

Answer 6

Decrease if the Na/K pump performance trigger seizure

Na/K blocker STDN (strophanthdin)

Chloride needs lots of potassium in the cell to be able to pump it out

Slide 18 Lectures 3/4

Question 7

What separation do charged is needed to change membrane voltage?

Answer 7

Not a lot needed

Example slide 19 Lectures 3/4

Question 8

What are Na+ and K+ behaviours on the membrane?

Answer 8

High external Na+ and low internal Na+ means the eq potential for Na+ is very positive and that Na+ ions tend to flow inward at physiological potentials

High internal K+ and low external K+ means that eq potential for K+ is very negative and K+ ions tend to flow outward at physiological potentials

Slide 36 Lectures 3/4

Question 9

What is the log of 10?
What is the log of 0.1?
What is the log of 1?

Answer 9

Log 10 = 1
Log 0.1 = -1
Log 1 = 0

Question 10

How can an ionic gradient and a semi-permeable membrane generate membrane potential?

Answer 10

1. Ionic gradient results in a net diffusion of ions toward the compartment of lower concentration
2. Ions will tend to flow toward a compartment that has an opposite charge

Question 11

How is the resting potential of a cell determined?

Answer 11

Resting potential of a cell is determined by the relative proportion of different types of ion channels that are open, together with the value of their equilibrium (Nernst) potentials

In a resting cell, only K+ channels are present, K+ ions are in equilibrium and Vm=EK

Question 12

How can we calculate the potential of a cell when there is more than one type of channel open?

Answer 12

The Goldman equation enables us to calculate how the contribution of multiple currents determines the resting membrane potential

This equation shows us we can change the resting membrane potential for a cell by changing the gradient for a given ion or changing the relative permeability for an ion

Slides 26-27 Lectures 3/4

Question 13

What is potential difference, current, and resistance?

Answer 13

Potential difference (E)- potential to do work between two points and is measured in volts (V)
Current (I)- net flow of charge from one point to the other measured in ampere (A)
Resistance (R)- resistance to the movement of current measured in ohms (Ω)

Question 14

How does the plasma membrane work as a capacitor?

Answer 14

Capacitance- ability of a system to store an electric charge

The nonconducting phospholipid bilayer separates the cytoplasm and extracellular fluid, both are highly conductive

Presence of thin layer of opposing charges on each side give rise to the electrical potential difference across membrane

Question 15

What is the equation for the electrical potential difference or voltage across a capacitor?

Answer 15

V=Q/C

Q= net charge (coulombs)
C= capacitance (farads)

Question 16

What are the ohms law equations with membrane potentials?

Answer 16

Vm= ik x rk

ik = Vm / rk

ik = Vm x γk

i= current
V= voltage
R= resistance
g(γ)= conductance

Slide 32-33 Lectures 3/4

Question 17

How do you find the current for potassium?

Answer 17

There is a K+ concentration gradient and a chemical force driving K+ across the membrane

ik = γk x (Vm-Ek)

(Vm-Ek) is the electrochemical driving force
Ek is the electromotive force

Slides 34-35 Lectures 3/4

Question 18

How can channel membranes be drawn as circuits?

Answer 18

Slide 37-38 Lectures 3/4

Question 19

What would happen to Ena if we increased or decreased the external concentrations of NaCl or altered the number of leak channels?

Answer 19

Increase potassium leak channels, more hyperpolarized

Increased sodium leak channels, more depolarized

Question 20

At rest is the cell at equilibrium?

Answer 20

No

Question 21

What are the 2 techniques for electrophysiological measurement?

Answer 21

Current clamp- measurement of cell voltage while controlling applied current
Voltage clamp- measurement of cell currents while controlling cell voltage

Compare the 2 on slide 6 Lecture 5

Question 22

What is capacitative current?

3 scenarios?

Answer 22

Only flows while Vm is changing
When voltage across a capacitor changes, the capacitor either gains or loses charge. This movement of charge onto or off the capacitor is an electrical (capacitative) current

A. Open, capacitor maintains its charge
B. Closed, capacitor discharges through resistor and voltage difference between the circuit points labelled in and out decays from V0 to final value of zero
C. Voltage decay follows an exponential time course, time required for voltage to fall to 37% of initial value is time constant (τ)
τ=Rm • Cm

Slide 4 Lecture 5

Question 23

How does a typical voltage clamp experiment work?

Answer 23

Oocyte 2 electrode voltage clamp- one extends monitors Vm and the other passes enough current (Im) through the membrane to clamp Vm to a predetermined command voltage (Vcommand)

Voltage clamp method of measuring ionic current- 2 situations on slide 7 Lecture 5 ones hyperpolarization ones depolarization

Question 24

How to measure Im by voltage clamping?

Answer 24

Voltage-clamping- to deduce properties of ion channels
An amplifier injects current into the cell to set the membrane voltage, device measures total current required to clamp Vm to desired value
When there is a difference from the intended voltage, feedback amplifier rapidly I hefts opposing current to maintain a constant Vm (so Vm is clamped)

If channel opens at clamped step voltage, the amplifier Injects current to keep Vm constant (that current is equal but opposite in sign)

Im = Ic + Ix
Im= Ic+Gx(Vm-Ex)
Ic=capacitative current
Ix= ionic current
Slide 8 Lecture 5

Question 25

How do we answer the question raised by voltage clamp channels of how many channels are involved in the production of a macroscopic current?
What are it’s types?

Answer 25

Patch-clamp configuration- high resistance seal between pipette glass and cell membrane allows measurement of activity of indivisible channels

Cell attached- maintains intracellular environment intact
Whole-cell- all membrane channels together (enables recording of all currents on cell membrane)
Inside-out- gain intracellular access (variable) (cytoplasmic side is facing bath solution)
Out-side out- gain extracellular access at single channel level (variable) (extracellular side of channel is facing out into bath solution)

Question 26

What is cell attached configuration?

What is the pipette solution?

Answer 26

Slide 12 Lecture 5
Channel remains on cell surface
Cells cytoplasm remind intact
Pipette solution- extracellular

Suction is applied to quiet electrical noise to see currents

Most used as it is step 1 for any configuration

Question 27

What is whole-cell configuration?

What is the pipetted solution?

Answer 27

Slide 13 and slide 17 Lecture 5
More suction used that creates direct access from pipette to cytoplasm
Pipette solution is intracellular and is in contact with cytoplasm

Records all currents on cell membrane (sucking from environment into cell)

Question 28

What is inside-out configuration?

What is the pipetted solution?

Answer 28

Slide 15 Lecture 5
Pulled membrane breaks away and forms loop (vesicle) in pipette
Pipette solution- extracellular
Inside-out excised patch- cytoplasmic side of channel is facing into bath solution

Useful when studying intracellular modulation of ion channels (phosphorylation, ligands, etc)

Question 29

What is outside-out configuration?

What is it’s pipetted solution?

Answer 29

Slide 14 and slide 18 Lecture 5
Strong suction breaks membrane and forms a U in pipette tip
Pipette solution- intracellular

Outside out excised patch- extracellular side of channel is facing out into bath

Used when studying activation of neurotransmitter receptors

Question 30

What is open-shut gating of ion channel?

Answer 30

Single channel current through a muscle nicotinic receptor
Records only one channels from membrane (outside out config)

The single channel conductance (γ) for a given channel is an inherent characteristic (conductance is known)

Question 31

What does a negative current for anion mean for the ion in cell?

Answer 31

Means ion is leaving cell

Slide 19 Lecture 5

Question 32

What is the current-voltage (IV) relationship?

Answer 32

Slide 19 Lecture 5

Question 33

What is macroscopic and microscopic currents?

Answer 33

Macroscopic current- current given by all ion channels in cell
Microscopic current- current given by single channel

Single channel currents sum to produce macroscopic currents
Slide 20-22 Lecture 5