MthK Gating Explored with a High Affinity Quaternary-Ammonium Blocker

Abstract

The bacterial potassium channel MthK is activated by Ca2+ binding to intracellular RCK-domains. The channel is homologous to the K+ conductive pore and C-terminal Ca2+ binding domains of the eukaryotic BK channel but lacks the N-terminal voltage-sensing domain. A low resolution x-ray crystal structure showed the MthK pore in a proposed open conformation, consistent with the presence of Ca2+ in the crystals (Jiang et al. Nature, 2002). It is widely assumed that the closed state of the channel involves an intracellular constriction of pore-helices as seen in the KcsA crystal structure (Doyle et al. Science, 1998; Jiang et al. Nature, 2002). However, several ligand-gated K+ channels, including the BK channel, have been proposed to use the selectivity-filter near the extracellular side and not an intracellular constriction as the conduction gate (Wilkens and Aldrich, JGP, 2006). We tested the presence of an intracellular gate in MthK by using a quaternary-ammonium (QA) blocker, bbTBA. QA blockers bind within the aqueous vestibulethat lies between the proposed intracellular gate and the selectivity-filter 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). We measured the bbTBA binding affinity to the open channel and have studied the state-dependence of channel block using single-channel recording in artificial bilayers. Preliminary data indicate the channel can close while the blocker remains bound. Further experiments involving macroscopic recordings may distinguish whether the blocker is trapped inside the closed channel or has state-independent binding.