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.
Ashmole
I
I
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Vavoulis
D V
DV
Stansfeld
P J
PJ
Mehta
Puja R
PR
Feng
J F
JF
Sutcliffe
M J
MJ
Stanfield
P R
PR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D024683:Potassium Channels, Tandem Pore Domain / Q000502:physiology*
C498655:KCNK9 protein, human; D024683:Potassium Channels, Tandem Pore Domain
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