Question

1. You lowered a patch-clamp onto a single ion channel. How would you identify what ion species it conducts?

2. You lowered a patch-clamp onto a single ion channel, which was created from RNA that you modified from RNA for a voltage-gated sodium channel gene. Name at least one feature of that ion channel you could identify that might tell you something useful about what the piece of ion channel you modified, might do.

Answer 1

Answer;

The Patch-clamp technique is a versatile electrophysiological tool for understanding ion channel behavior.

Every cell expresses ion channels, but the most common cells to study with patch-clamp techniques include neurons, muscle fibers, cardiomyocytes, and oocytes overexpressing single ion channels. To evaluate single ion channel conductance, a microelectrode forms a high resistance seal with the cellular membrane, and a patch of cell membrane containing the ion channel of interest is removed.

1)

Patch-clamp electrophysiology is the first and most widely used experimental method sufficiently sensitive to permit the observation of single molecules1,2. In addition to this exquisite sensitivity, it has vastly expanded the biological preparations amenable to electrophysiologic recording and also has allowed the observation of ion channels in intact membranes. First, because both voltage clamping and current recording are accomplished with the same electrode, it can be used to record signals across small cells or membranes patches. The technique revealed that ion channels are not restricted to excitable membranes of frog muscles, eel electroplaques, or squid giant axons3,4, but rather that they represent ubiquitous fixtures of transmembrane signaling mechanisms and are intrinsic to all cellular membrane types of uni- or multicellular organisms, and also to intracellular membranes. Importantly, the capability to record transmembrane currents by simply attaching a glass pipette to an intact cell provided the unprecedented opportunity to record activity from ion channels in their native undisrupted membranes. Thus, the cell attached patch-clamp technique, which is described in this protocol, permits monitoring the activity of ion channels continuously for tens of min or longer in their native environment.

Under normal thermal fluctuations, all proteins, including ion channel proteins, undergo structural changes over a broad time scale, with the fastest and most frequent rearrangements represented most likely by side-chain movements and much slower, less frequent changes represented by the repositioning of entire domains or subunits, or in some cases by post translational modifications or protein-protein interactions5,6. Observing long periods of activity generated by one molecule can help to understand the functional dynamics of ion channels in intact physiological membranes and provides valuable information about the operational mechanism of the molecule observed.

Patch clamp identify ion species it conducts like in brain.

patch electrode is attached to the cell but the membrane is not broken, has been widely used for recording single channel currents, for recording the summed current of many single channels in a patch of membrane, and for recording spontaneous cell firing activity. In this review I will use theory and new experimental data from neurons in hippocampal slices to discuss three uses of cell-attached recording: 1) the recording of resting and synaptic potentials, 2) the recording of spontaneous cell firing activity, and 3) the stimulation of the attached cell.

Example how patch clamp give variation.;

Cell-attached Inside-out Pipette Withdraw Pipette Gigaseal Cell Suction Zap Gigaseal Withdraw Pipette Perforated Whole-cell Outside-out

Diagram showing variations of the patch clamp technique

Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of individual ion channels in the section of membrane attached to the electrode.

2)

patch-clamp techniques include neurons, muscle fibers, cardiomyocytes, and oocytes overexpressing single ion channels. To evaluate single ion channel conductance, a microelectrode forms a high resistance seal with the cellular membrane, and a patch of cell membrane containing the ion channel of interest is removed. Alternatively, while the microelectrode is sealed to the cell membrane, this small patch can be ruptured giving the electrode electrical access to the whole cell. Voltage is then applied, forming a voltage clamp, and membrane current is measured. Current clamp can also be used to measure changes in membrane voltage called membrane potential. Voltage or current change within cell membranes can be altered by applying compounds to block or open channels. These techniques enable researchers to understand how ion channels behave both in normal and disease states and how different drugs, ions, or other analytes can modify these conditions.

Features of ion channels;

ion channel expression is relatively simple, greatly enhances the speed and efficiency with which high quality macroscopic current data can be collected, and makes it possible to easily and reliably record single channel currents in a mammalian cell heterologous expression system.

Whereas we demonstrate the ability of this system to control expression levels of voltage-gated K+ channels, it should be applicable to all other channel types that express well in mammalian expression systems.