Introduction and Experimental Traces

Question 1

give 3 ways in which ion channels are categorised and examples of each

Answer 1

1. ion selectivity (Na, K, Ca etc)
2. gating properties (voltage gated, ligand gated)
3. molecular structure (amino acid homology, eg. Na_v1.1-1.9

Question 2

1. Give the structure of K_v
2. Give the structure of Na_v
3. what does Ca_v have a similar structure to?
4. what is the shape of the current through K_ir channels?

Answer 2

1. 4 proteins come together to form a functional channel. Each protein has 6 TM domains. the S4 region forms the voltage sensing region, and the 4 pore regions between S5 and S6 come together to form the ion channel pore
2. 1 protein forms a functional channel. 24 TM domains (4 sets of 6); the S4 region in each set of 6 forms the voltage sensing regions; the pore regions are found between S5 and S6 of each set of 6; single TM domain containing beta subunits regulate the alpha subunit
3. Na_v
4. Inwardly rectifying currents

Question 3

1. give the equation which states what current is influenced by
2. for N, Vm and Po, state what each is dependent upon (for Po, give examples for each factor)

Answer 3

1. I=N.Po.g.(Vm-Ei)

where N is number of channels, Po is open probability, g is single channel conductance, and Vm-Ei is driving force

2. N is dependent on trafficking of the channel to the surface

Po is dependent on various factors sucg as voltage (Nav), phosphorylation (TRP), g proteins (TRP channels in drosophila photoreceptors) Calcium (CRAC channels) etc

Vm is dependent on the inhibition or activation of other channels

Question 4

1. Under physiological conditions, give the values of E_k, E_Na, and Vm
2. what do these values imply in terms of membrane permeability?

Answer 4

1. E_k = -90mVE_Na = +60mVVm = ~-45mV
2. Implies that the membrane is more permeable to K than Na

Question 5

Patch Clamp and Two Electrode Voltage Clamp Techniques

1. what are these techniques used for
2. what does the apparatus do?
3. What type of cells is the patch clamp technique used for?
4. What type of cells is the two electrode technique used for?

Answer 5

1. to measure the current at a particular Vm
2. the interior of the pipette is filled with solution matching ionic components of the cytoplasm. A chloride silver wire is placed in contact with this solution and conducts electrical current allowing the Vm to be manipulated
3. small cells; the electrode sets Vm and measures current
4. large cells; one electrode sets Vm and the second measures current.

Question 6

Positive and Negative Currents

1. in terms of cations, in which direction of ion movement causes positive and negative currents?
2. in terms of anions, in which direction if ion movement causes positive and negative currents?

Answer 6

1. Positive currents - outward movementNegative currents - inward movement
2. Positive currents - inward movementNegative currents - outward movement

Question 7

1. When a blocker is used, and a current moves closer to zero, what is implied?
2. why may a blocker not reduce a current completely to zero? (2)
3. why do older blockers tend to be less specific to newer blockers?
4. give 2 examples of Na_v blockers
5. give 2 examples of K_v blockers
6. give 2 examples of Kir blockers
7. give 2 examples of TRPV blockers
8. give an example of a CLC blocker

Answer 7

1. that the channel that governs the current has been inhibited
2. a submaximal concentration of the blocker has been used, or that some channels are insensitive to the blocker
3. because they have been tested on more things therefore have been found to inhibit many channels. This may also be true for newer blockers, however their effects are yet unknown
4. tetrodotoxin; ProTx-II
5. TEA; barium
6. Barium, quinidine
7. gadolinium, ruthenium red
8. DIDS