Structural Basis of a Kv7.1 Potassium Channel Gating Module: Studies of the Intracellular C-Terminal Domain in Complex with Calmodulin

Abstract

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation

“…To place the data of this report into physical perspective, Fig. 1 shows a tentative disposition of CaM on Kv7.2 forming a ring under the pore that complies with numerous restrictions from different studies (Mruk et al, 2012;Xu et al, 2013;Alaimo et al, 2014;Sachyani et al, 2014) (see Materials and Methods). One important feature derived from the 3D structure of the Kv7.1 [ABCD/CaM] complex is that the helix D coiled-coil is separated from the membrane by the CaM ring (Sachyani et al, 2014) (Fig.…”

“…Helix A of Kv7.2 presents a marked preference for fetching the C-lobe, whereas helix B anchors more favorably to the N-lobe and these features are present on the crystallographic Kv7.1 [AB/CaM] complex (Sachyani et al, 2014). To place the data of this report into physical perspective, Fig.…”

“…Inspired by this precedent, (Sachyani et al, 2014), the [B/CaM] Kv7.4 complex (Xu et al, 2013), helix D Kv7.4 and Kv7.1 tetrameric bundles (Howard et al, 2007;Wiener et al, 2008), and the model derived from internal TEA-CaM blockade (Mruk et al, 2012). The disposition of helix C and the CD linker are unknown (dashed lines).…”

“…These data reinforce the initial conclusion, based on yeast two-hybrid interaction trap and pulldown assays, that helices CD do not contribute directly to CaM binding (Yus-Nájera et al, 2002). Similarly, a role of CD helices has been discarded for CaM binding to Kv7.4 and Kv7.1 channels (Xu et al, 2013;Sachyani et al, 2014). In contrast, an increased FRET index compared with that of AB was observed between CFP-CaM and YFP-ABCD (Fig.…”

“…CaM regulatory mechanisms that change the gating behavior proceed through an effect on sensitivity to PIP 2 (Kosenko et al, 2012;Kosenko and Hoshi, 2013;Zaydman et al, 2013;Kang et al, 2014). The interlinker between helices A and B is not essential for function, although it plays a critical role in PIP 2 regulation for Kv7.3, but not in Kv7.1 and Kv7.2 channels (Hernandez et al, 2008;Aivar et al, 2012;Sachyani et al, 2014).…”