Influence Of The Kcsa C-terminal Domain In The Coupling Between Activation And Inactivation Gates

Abstract

Truncation of KcsA C-terminal domain (CTD) been reported to impair ion channel activity 1. However, we have shown that a KcsA lacking the CTD (KcsA-ΔCTD) is capable of catalyzing pH-dependent rubidium influxes 2.To investigate the functional and structural roles of KcsA CTD in channel gating, we have studied pH-dependent structural changes of the activation gate by EPR and Fluorescence spectroscopy in full length (FL) and ΔCTD KcsA. Proton-dependent macroscopic currents of KcsA-ΔCTD inactivated faster and deeper when compared to the FL channel. Additionally, single channel analysis showed that at steady state KcsA-ΔCTD has an open probability Po not higher than ∼ 0.001, about one order of magnitude lower than FL-KcsA. Recently, by solving a family of KcsA-ΔCTD open structures we have proposed the mechanism by which the activation gate is allosterically coupled to the selectivity filter. As a result, we have hypothesized that a larger opening at the activation gate in KcsA-ΔCTD is directly correlated with an enhancement in the rate of inactivation. Distances estimated by fluorescence resonance energy transference (FRET) indicates that KcsA-ΔCTD activation gate opened to a larger extent than that in FL-KcsA, thus strengthening the coupling between activation and the collapse of the selectivity filter. Our x-ray structures of closed and open FL-KcsA in addition to the KcsA-ΔCTD in the open conformation are in agreement with a mechanistic model where the larger the opening at the activation gate the deeper inactivation at the selectivity filter.1 F. I. Valiyaveetil, M. Sekedat, T. W. Muir et al., Angew Chem Int Ed Engl 43 (19), 2504 (2004).2 D. M. Cortes, L. G. Cuello, and E. Perozo, The Journal of general physiology 117 (2), 165 (2001).