Voltage clamp

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential (voltage) to a desired value by adding the necessary current. This 'clamps' the cell membrane at a desired constant voltage, allowing the voltage clamp to record what currents are delivered. Because the currents applied to the cell must be equal to (and opposite in charge to) the current going across the cell membrane at the set voltage, the recorded currents indicate how the cell reacts to changes in membrane potential. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage-gated. The voltage clamp allows the membrane voltage to be manipulated independently of the ionic currents, allowing the current-voltage relationships of membrane channels to be studied. The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential (voltage) to a desired value by adding the necessary current. This 'clamps' the cell membrane at a desired constant voltage, allowing the voltage clamp to record what currents are delivered. Because the currents applied to the cell must be equal to (and opposite in charge to) the current going across the cell membrane at the set voltage, the recorded currents indicate how the cell reacts to changes in membrane potential. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage-gated. The voltage clamp allows the membrane voltage to be manipulated independently of the ionic currents, allowing the current-voltage relationships of membrane channels to be studied. The concept of the voltage clamp is attributed to Kenneth Cole and George Marmont in the spring of 1947. They inserted an internal electrode into the giant axon of a squid and began to apply a current. Cole discovered that it was possible to use two electrodes and a feedback circuit to keep the cell's membrane potential at a level set by the experimenter. Cole developed the voltage clamp technique before the era of microelectrodes, so his two electrodes consisted of fine wires twisted around an insulating rod. Because this type of electrode could be inserted into only the largest cells, early electrophysiological experiments were conducted almost exclusively on squid axons. Squids squirt jets of water when they need to move quickly, as when escaping a predator. To make this escape as fast as possible, they have an axon that can reach 1 mm in diameter (signals propagate more quickly down large axons). The squid giant axon was the first preparation that could be used to voltage clamp a transmembrane current, and it was the basis of Hodgkin and Huxley's pioneering experiments on the properties of the action potential. Alan Hodgkin realized that, to understand ion flux across the membrane, it was necessary to eliminate differences in membrane potential. Using experiments with the voltage clamp, Hodgkin and Andrew Huxley published 5 papers in the summer of 1952 describing how ionic currents give rise to the action potential. The final paper proposed the Hodgkin–Huxley model which mathematically describes the action potential. The use of voltage clamps in their experiments to study and model the action potential in detail has laid the foundation for electrophysiology; for which they shared the 1963 Nobel Prize in Physiology or Medicine. The voltage clamp is a current generator. Transmembrane voltage is recorded through a 'voltage electrode', relative to ground, and a 'current electrode' passes current into the cell. The experimenter sets a 'holding voltage', or 'command potential', and the voltage clamp uses negative feedback to maintain the cell at this voltage. The electrodes are connected to an amplifier, which measures membrane potential and feeds the signal into a feedback amplifier. This amplifier also gets an input from the signal generator that determines the command potential, and it subtracts the membrane potential from the command potential (Vcommand – Vm), magnifies any difference, and sends an output to the current electrode. Whenever the cell deviates from the holding voltage, the operational amplifier generates an 'error signal', that is the difference between the command potential and the actual voltage of the cell. The feedback circuit passes current into the cell to reduce the error signal to zero. Thus, the clamp circuit produces a current equal and opposite to the ionic current. The two-electrode voltage clamp (TEVC) technique is used to study properties of membrane proteins, especially ion channels. Researchers use this method most commonly to investigate membrane structures expressed in Xenopus oocytes. The large size of these oocytes allows for easy handling and manipulability. The TEVC method utilizes two low-resistance pipettes, one sensing voltage and the other injecting current. The microelectrodes are filled with conductive solution and inserted into the cell to artificially control membrane potential. The membrane acts as a dielectric as well as a resistor, while the fluids on either side of the membrane function as capacitors. The microelectrodes compare the membrane potential against a command voltage, giving an accurate reproduction of the currents flowing across the membrane. Current readings can be used to analyze the electrical response of the cell to different applications. This technique is favored over single-microelectrode clamp or other voltage clamp techniques when conditions call for resolving large currents. The high current-passing capacity of the two-electrode clamp makes it possible to clamp large currents that are impossible to control with single-electrode patch techniques. The two-electrode system is also desirable for its fast clamp settling time and low noise. However, TEVC is limited in use with regard to cell size. It is effective in larger-diameter oocytes, but more difficult to use with small cells. Additionally, TEVC method is limited in that the transmitter of current must be contained in the pipette. It is not possible to manipulate the intracellular fluid while clamping, which is possible using patch clamp techniques. Another disadvantage involves 'space clamp' issues. Cole's voltage clamp used a long wire that clamped the squid axon uniformly along its entire length. TEVC microelectrodes can provide only a spatial point source of current that may not uniformly affect all parts of an irregularly shaped cell.