Part 1: CW/WS

Question 1

On-cell patch:

Answer 1

• Measures indirect effect of extracellular solutes on channels within membrane patch on intact cell (glass pipette used)
• Measuring activity

Question 2

Inside-out patch:

Answer 2

• Separating out a single channel
• Pull it out of vesicle, wash etc.
• Less physiological and more controlled

Question 3

How is channel activity determined in patch clamp experiments?

Answer 3

• Clamp established
• Current provided
• Voltage steadied
• Measuring amount of current required to maintain this voltage -> plot current against voltage

Question 4

How is the Nernst equation applied?

Answer 4

• Calculates the voltage at which there is no ‘electrochemical driving force’ for the passive selective movement of ion species across a membrane at a given concentration gradient
• As such, predicting voltage at which no current will occur
• This is carried out for each ion in bath

Question 5

Open probability of a channel:

Answer 5

• Open time / total time
• Dependent on a ligand-gating or voltage

Question 6

Ohm’s law:

Answer 6

• V = I*R
• Describes relationship between current (I in Amperes), voltage (V in Volts) and resistance (R in Ohms)
• Relationship between I and V is thus linear with gradient of R

Question 7

How are microelectrodes made?

Answer 7

• Glass capillary is fixed in an electrode puller
• Current run through heating coil -> heat melts glass, gravity pulls apart the two halves

Question 8

Basic patch clamp rig:

Answer 8

• Current measuring device (amplifier)
• Microscope
• Micromanipulator

Question 9

How is reversal potential calculated?

Answer 9

• I-V plot
• mV on X and pA on Y
• Find x-intercept for each bath

Question 10

Converting degrees to kelvin

Answer 10

• +273

Question 11

S.cerevisiae high affinity Calcium uptake system:

Answer 11

• Cch1(homolog of a subunit of L-type VGCC)
• Mid1 (TM protein which interacts with Cch1)
• Ecm7
• HACS is important for growth when extracellular calcium is very limited
• Conversely, molecular identity of LACS is unclear (Fig1?)

Question 12

S.cerevisiae: I-C release mechanisms

Answer 12

• TRPY1/Yvc1 (TRP homolog) -> vacuolar membrane, stretch activated

Question 13

Role of Vcx1 and Pmc1 in S.cerevisiae:

Answer 13

• Vcx1: Low affinity high capacity Ca2+/H+ antiporter in vacuolar membrane (restoring basal after peak)
• Pcm1: Ca2+-ATPase in vacuolar membrane (maintaining basal levels prior to signalling events)