Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating.
In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.
Choi
Kee-Hyun
KH
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Tantama
Mathew
M
Licht
Stuart
S
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000495:Allosteric Site; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D015640:Ion Channel Gating; D007700:Kinetics; D016013:Likelihood Functions; D051379:Mice; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry*; D011487:Protein Conformation; D013816:Thermodynamics
D024661:Potassium Channels, Inwardly Rectifying; D000255:Adenosine Triphosphate