BK-type calcium-activated potassium channels serve as cytoplasmic Ca2+ detectors that can rapidly respond to and modulate transmembrane voltage. These channels are critical in controlling action potential firing in neurons as well as smooth muscle contractility, and consequently, dysfunction of BK channels is associated with movement disorders and epilepsy in humans, and with hypertension in mouse models. An understanding of the structural basis for gating in BK channels thus has direct relevance to human disease, and may ultimately lead to novel therapeutic interventions. In the absence of a 3-D structure for the BK channel, we seek to gain structural insights toward mechanisms of Ca2+-dependent channel activation through an understanding of the prokaryotic Ca2+-gated K channels MthK and TvoK, which are amenable to both functional and structural study. BK, MthK, and TvoK all contain RCK domains that immediately follow their pore-lining helices. The common molecular architecture shared by BK, MthK, and TvoK suggests that understanding the conformational dynamics of the prokaryotic relatives will provide mechanistic insight relevant to BK channels. Our approach toward gaining these novel insights will be multidisciplinary, using electrophysiology to assay function and NMR spectroscopy to probe structure. The specific insights we seek to gain concern 1) the relation between Ca2+-dependent movements at the RCK subunit interfaces and channel gating and 2) the [Ca2+]-dependence of individual residue movements in RCK subunits. Our proposed research over the coming two-year period is focused on two aims: 1) To determine the energetic contributions of intersubunit bonds to Ca2+-dependent gating in MthK. We will test the energetic contributions of salt bridges and hydrogen bonds at RCK domain subunit interfaces by targeting the individual sidechain components of these interactions with mutagenesis, and assaying the mutation effects on gating using single-channel electrophysiology. 2) To identify Ca2+-dependent intermediate structural conformations in an RCK domain. Here we will analyze the relation between structural movements and [Ca2+] at the atomic scale using solution NMR spectroscopy, by measuring chemical shift perturbations of individual residues as a function of [Ca2+]. These experiments will enable the resolution of conformational steps in the Ca2+-dependent gating pathway that cannot be resolved using electrophysiological or X-ray experiments.
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