Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-Helix Complex Provides Insight into M Current Modulation

Abstract

Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M-currents and cardiac and auditory IKS currents, and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca2+/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca2+/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca2+/CaM:Kv7.4 B segment complex shows that Ca2+/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca2+/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca2+/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.

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

“…Neither our new FRET data, nor previous studies using yeast two hybrid assays, or studies on Kv7.1 or Kv7.4, have exposed any hint of direct CaM interaction with helices CD (Yus-Nájera et al, 2002;Howard et al, 2007;Wiener et al, 2008;Xu et al, 2013). In addition, small angle X-ray scattering of the related Kv7.1 subunit (Sachyani et al, 2014) The CaM N-lobe has a significant preference to anchor to helix B, whereas the C-lobe binds more favorably to the IQ site located in helix A .…”

“…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.…”

“…1C). Thus, similar to Kv7.1 or Kv7.4 [AB/ CaM] complexes studied in vitro (Wiener et al, 2008;Xu et al, 2013), the Kv7.2 [AB/CaM] complex adopts a 1:1 stoichiometry in solution.…”

“…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.…”