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© The Rockefeller University Press, 0022-1295/1998//333/ $5.00
Journal of General Physiology, Volume 112, Number 3, 1998

Article

Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

Stefan Trapp, Peter Proks, Stephen J. Tucker, and Frances M. Ashcroft

From the University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2{Delta}C26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2{Delta}C26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2{Delta}C26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 µM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

Key Words: ATP-sensitive K+ channel • cysteine • Kir6.2 • SUR1 • gating

Address correspondence to Frances M. Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. Fax: 44-1865-272469; E-mail: frances.ashcroft{at}physiol.ox.ac.uk

Abbreviations: wt, wild type


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S. Trapp, S. J. Tucker, and F. M. Ashcroft
Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification
J. Gen. Physiol., September 1, 1998; 112(3): 325 - 332.
[Abstract] [Full Text] [PDF]


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