The response of the tandem pore potassium channel TASK-3 (K(2P)9.1) to voltage: gating at the cytoplasmic mouth.

Although the tandem pore potassium channel TASK-3 is thought to open and shut at its selectivity filter in response to changes of extracellular pH, it is currently unknown whether the channel also shows gating at its inner, cytoplasmic mouth through movements of membrane helices M2 and M4. We used two electrode voltage clamp and single channel recording to show that TASK-3 responds to voltage in a way that reveals such gating. In wild-type channels, P(open) was very low at negative voltages, but increased with depolarisation. The effect of voltage was relatively weak and the gating charge small, 0.17. Mutants A237T (in M4) and N133A (in M2) increased P(open) at a given voltage, increasing mean open time and the number of openings per burst. In addition, the relationship between P(open) and voltage was shifted to less positive voltages. Mutation of putative hinge glycines (G117A, G231A), residues that are conserved throughout the tandem pore channel family, reduced P(open) at a given voltage, shifting the relationship with voltage to a more positive potential range. None of these mutants substantially affected the response of the channel to extracellular acidification. We have used the results from single channel recording to develop a simple kinetic model to show how gating occurs through two classes of conformation change, with two routes out of the open state, as expected if gating occurs both at the selectivity filter and at its cytoplasmic mouth.

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1: contributions of the pore domains.

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels non-selective. We interpret the effects of these--and of other mutations--in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter.