KcsA Gating Explored with Quaternary-Ammonium Blockers

Abstract

The bacterial potassium channel KcsA, an archetypal K+ channel pore, is proposed to close at an intracellular constriction. The inner helices form a bundle crossing that separates the intracellular solution from a large, hydrated internal vestibule within the pore domain (Doyle et al. Science, 1998). This vestibule has been shown to be the receptor-site for open-channel blockers such as quaternary-ammonium ions in KcsA and other voltage-gated potassium channels (Armstrong and Hille, JGP, 1972; Holmgren et al. JGP, 1997; Zhou et al. Nature, 2001; Lenaeus et al. NSMB, 2005; Yohannan et al. JMB, 2007). Since KcsA is gated by intracellular protons, it is predicted that pH will dramatically alter the accessibility of channel blockers to the vestibule. We are exploring the state-dependence of channel block by quaternary-ammonium ions using steady-state single channel recording of the non-inactivating KcsA E71A channel (Cordero-Morales et al. NSMB, 2006). Preliminary results indicate a profound state-dependence, with TBA blocking kinetics and percent block changing dramatically as a function of channel open probability. We will compare these results with blocking data for a pH-insensitive KcsA mutant we previously reported (Thompson et al. PNAS, 2008). These results demonstrate that the pH-sensor of KcsA operates to gate ion access to the vestibule.