The Effect of D2O on the Inactivation Kinetics and Recovery from Slow Inactivation of Shaker-IR K+ Channels

Abstract

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation which is stabilized by a multipoint hydrogen-bond network behind at the selectivity filter in eukaryotic KV channels. The selectivity filter is sterically locked in the inactive conformation by buried water molecules which prevent recovery from inactivation. To further test the critical role of these, structural water” molecules in the conformational stability of the selectivity filter we studied the effects of substituting water for heavy water (deuterium oxide, D2O in the solutions) on various gating transitions in Shaker-IR channels having the following mutations and allowing slow inactivation at various rates: T449A, T449A/I470A and T449K/I470C. All channels were transiently expressed in tsA_201 cells and ionic currents were recorded in excised inside-out patches. 2.0-s-long depolarizing pulses from a holding potential of −120 mV to +50 mV were applied to measure the current through the channels. The extracellular application of D2O dramatically slowed entry into the inactivated state, the inactivation time constats were τi,D2O = 189±48 ms (n=5) (T449A, τi,H2O = 72±13 (n=5)), τi,D2O = 418±63 ms (n=4) (T449A/I470A, τi,H2O = 247±90 (n=5)), τi,D2O = 60±21 ms (n=6) (T449K/I470C, τi,H2O = 31±15 ms (n=10)), respectively. In contrast, applying D2O from the intracellular side did not change the inactivation kinetics. The kinetics of recovery from slow inactivation was also slowed down in the D2O environment. Our macroscopic current measurements confirm that water molecules may have access to the region behind the selctivity filter only from the external solution and the binding of these ‘hidden’ molecules slows the development of inactivation and that of the recovery process.