Question

You are studying a ligand-gated ion channel. The ligand is a neurotransmitter. Upon binding the neurotransmitter, the ion channel opens and Na+ flows into the cell. The receptor is found on the dendrites of frog neurons. You are going to use one of the patch-clamp variations to study the effect of (1) the neurotransmitter and (2) second (and separately) the effect of an antagonist. Explain the patch-clamp technique variation you would use and why. Explain how you will study the effect of neurotransmitter and the antagonist. Diagram what this experiment would look like (the clamp, the ion channel, and where you put the neurotransmitter or antagonist to test these molecules). Explain, what would be the result, at the cellular level, if a neuron was exposed to the neurotransmitter or antagonist. Finally, draw an action potential graph showing the effects of the neurotransmitter or antagonist (draw a normal action potential and then draw the effect with the neurotransmitter present). On a second graph, draw a normal action potential and then the effect of the antagonist.

Answer 1

1) I’d use the loose patch technique since I’m not interested in studying the internal changes produced by the activation of the ligand-gated ion channel. This technique consists in approaching the micropipette with the electrode (with a solution of isotonic buffer, ions and the ligand used in the assay) to the membrane, touching it but not sucking (therefore, there isn’t a gigaseal (a patch of membrane with the Ω shape) like in the other variants). If I was interested in the internal changes in the cell I’d use cell-attached patch technique.

2) I’d fill the pipette solution with the same mix but I’d prepare 3 treatments (nothing; with neurotransmitter; with antagonist) and fill micropipettes with electrodes with those solutions and start “touching” the same place of the membrane (first with the “nothing” solution, second with the “neurotransmitter” and after a few minutes of resting with the “antagonist”) to obtain measures of the current.

3) Here’s the diagram, pardon me for my rudimentary drawing skills.

Touching but not Micropipettes with sucking the membrane electrode and solution Phospholipid bilayer Isotonic buffer, ions (Ca2+, Na+) nothing - lon channel Isotonic buffer, ions (Ca2+, Na+) NeuroT. Isotonic buffer, ions (Ca2+, Na+) Antagon.'

4) If the neuron is exposed to the neurotransmitter their membrane potential will polarize (it will become more positive or less negative) since Na+ will enter the cytoplasm and afterwards it will start an action potential but it will stabilize soon it’s membrane potential, but, if an antagonist binds to the ionic channel it will keep it open all the time therefore the Na+ influx will be continuous and the electrochemical gradient will disappear.