Help me understand voltage patch clamping

Question

Before I type my question it is important to know that I already tried looking this up on my own and could not find an answer because the answers are all in complicated physics terms and this topic is only half a page in a chapter on Action potential in my 1st year physiology textbook.

My textbook says:

This apparatus is used in the following way: The investigator decides which voltage to establish inside the nerve fiber. The electronic portion of the apparatus is then adjusted to the desired voltage, automatically injecting either positive or negative electricity through the current electrode at whatever rate is required to hold the voltage, as measured by the voltage electrode, at the level set by the operator. When the membrane potential is suddenly increased by this voltage clamp from −90 millivolts to zero, the voltage-gated sodium and potassium channels open and sodium and potassium ions begin to pour through the channels.

1: Does that mean that the change in membrane potential that I did resulted in changes in the conformational states of ion channels which in turn changed the membrane potential that I need to control ?

To counterbalance the effect of these ion movements on the desired setting of the intracellular voltage, electrical current is injected automatically through the current electrode of the voltage clamp to maintain the intracellular voltage at the required steady zero level.

2:Is this all still the step of setting the membrane potential to a certain specific value ?

To achieve this level, the current injected must be equal to but of opposite polarity to the net current flow through the membrane channels.

3: I'm not sure I understand this.

Finally, the investigator adjusts the concentrations of the ions to other than normal levels both inside and outside the nerve fiber and repeats the study. When sodium is the only permeant ion in the solutions inside and outside the squid axon, the voltage clamp measures current flow only through the sodium channels. When potassium is the only permeant ion, current flow only through the potassium channels is measured.

4: do they make sure that the flow is only from sodium channels by setting the membrane potential to the equilibrium potential of other ions or a potential between the equilibrium potential of the other ions (so greater than it) and the equilibrium potential of Na (and also less than it )?

Answer 1

I'll try to answer the sub questions 1-by-1

1. Does that mean that the change in membrane potential that I did resulted in changes in the conformational states of ion channels which in turn changed the membrane potential that I need to control ?

It's the other way around; your book is right - "When the membrane potential is suddenly increased by this voltage clamp from −90 millivolts to zero, the voltage-gated sodium and potassium channels open and sodium and potassium ions begin to pour through the channels". So the voltage step (depolarization) induced by the apparatus triggers voltage-operated channels to undergo conformational changes, this in turn results in additional current flowing through them. On the other hand, hyperpolarization of the potential from say -40 to -90 causes the neuron to close these channels, blocking current to flow. In other words; the patch clamp apparatus controls the membrane potential, the state of the ion channels has little to do with that, as the apparatus will automatically adjust the current injection.

2. Is this all still the step of setting the membrane potential to a certain specific value?

Yes.

3. "To achieve this level, the current injected must be equal to but of opposite polarity to the net current flow through the membrane channels." I'm not sure I understand this.

To set a certain stable potential across the membrane, the flow of current needs to be stable, otherwise it will fluctuate; Ohm's law tells us U = IxR. The resistance R is set by the channels. Let's consider that R is stable for the moment. In that case, you will set the membrane potential U by tightly controlling the current I. If I fluctuates, i.e., when the apparatus doesn't balance the flow of current through the membrane channels exactly and oppositely, a net current will start flowing and u will change. You don't want that to happen.

4. do they make sure that the flow is only from sodium channels by setting the membrane potential to the equilibrium potential of other ions or a potential between the equilibrium potential of the other ions (so greater than it) and the equilibrium potential of Na (and also less than it )?

This part has nothing to do with equilibrium potentials. They substitute negative ions simply by ions that are not membrane permeable, and the only positive ion available in the test tube is Na⁺. In that case, only Na⁺ can flow and you are sure that it is Na⁺ that is flowing. At least, this is what I make up of this text.