BIO254:Charge

Gating Charges
Voltage-gated ion channels open and close in response to changes in the electric environment of the membrane. This is achieved though a voltage sensor that detects voltage by use of key charged elements or “gating charges”. Changes in membrane potential cause motion of the gating charges thus inducing conformational changes in the whole protein and resulting in opening or closure of the channel. The opening event consists of positive charges moving outwardly while they move inwardly for closing the channels during repolarizations. The movement of these charges is detectable in voltage clamp as small current that precedes the ionic currents and is known as “gating current”. Their movement can also be detected using optical methods, where a fluorescent dye can be coupled to the outside of the channel and changes in fluorescence can be measured as the local environment changes due to charge movement. For many voltage-gated ion channels the charges are conserved positively charged amino acids and their identity has been studied extensively using mutagenesis and heterologous expression. Taken together, these studies indicate that most of the gating charges reside within the S4 segment of the channels.

For a great review see: '''The Voltage Sensor in Voltage-Dependent Ion Channels Francisco Bezanilla. Physiological Reviews, Vol. 80, No. 2, April 2000, pp. 555-592.'''

Experiments To Determine Gating Charges
The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. To determine which sections of the protein sequence is responsible for this voltage "switch-sensor," Aggarwal and MacKinnon (Neuron, 1996) created charge-neutralizing mutations on the first four positive charges from the N-terminus and the C-terminus. The gating charge response of C-terminus mutants was almost identical to that of wild-type channels; however, mutations induced on the N-terminus positive arginines resulted in channels that failed to open when the appropriate voltages were applied using the patch clamp method. Hence, their experiment shows that the movement of the NH2-terminal half but not the COOH-terminal half of the S4 segment underlies gating charge.

Movement of the Gating Charge
The end result of the gating charge is to open up the channel at a specified voltage. To do that the sensing element of the channel has to move in response to a membrane potential, and it has to transfer its movement to the pore gate, causing it to open. To understand the nature of the movement of the S4 segment--the part of that channel that seems most likely to contain the gating charges--groups sequentially mutated its residues to cysteines. By using thiol reacting agents in the extracellular space, these mutated cysteines allows for a determination of the location of each residue in different electrical states of the membrane. In addition, groups also attached flurophores to the sequential cysteines to study the distances moved by the various residues of the S4 segment using FRET. Although these indirect studies are not definitive, they all indicate that the S4 helix underoes a rotation upon depolarization. Some of the studies indicate that there might be addition movements, such as a coupled translocation on top of the rotation, or even, perhaps, movement by the surrounding helixes.

Once the S4 segement moves, it has to, somehow, convey its change to the tail ends of the S6 segment--the gate. Evidence is much more scarce as to how that happens. By changing the length of the S4-S5 linker, groups have shown that part of the channel to play a role, suggesting that the linker segment connects to the S6 tail. However, there is still a possibilty that the movement of the S4 segment causes more of a gross change within the protein, forcing the rest of the surrounding helixes to adapt to it change, which then lead to the opening of the gate.

Useful reviews: Horn, R. "A new twist in the saga of charge movement in voltage-dependent ion channels." Neuron. 2000 Mar;25(3):511-4 Horn, R. "Coupled movements in voltage-gated ion channels." J Gen Physiol. 2002 Oct;120(4):449-53