Structural Insights into the M-Channel Proximal C-Terminus/Calmodulin Complex

Abstract

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca(2+)concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca(2+) concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca(2+) CaM conformation even though four Ca(2+) ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties.

A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

“…It suggests a conformation of the apoCaM:KCNQ4 complex quite distinct from our Ca 2ϩloaded crystal structure ( Fig. 2) and others (45,46,50), of Ca 2ϩ / CaM in complex with the A and B domains of KCNQ1-4, and the cryo-EM structure of Ca 2ϩ /CaM:KCNQXem (47), which all show the B domain interacting with the N-lobe of CaM and the A domain embraced by the C-lobe. These results challenge our ini-tial supposition that apoCaM would be constitutively bound to the A domain and suggest a much more dynamic mechanism of Ca 2ϩ directing CaM interactions with KCNQ4 channels.…”

“…Contrary to these findings, the Ca 2ϩ /CaM: Q3AQ2B structure (PDB entry 5J03) found Ca 2ϩ coordinated in all four EF-hands formed in a crystallization buffer containing a stoichiometric ratio of [Ca 2ϩ ] to [CaM] of 125:1. Thus, we must admit the likely possibility that a greater stoichiometric excess of Ca 2ϩ to CaM than what we and others have used is required to fully load all "loadable" C-lobe EF-hands under crystallization conditions (45). A color plot of crystallographic B-factors of the crystal structure shows the relative level of disorder, and we observe that the EF-hands III and IV map the highest disorder ( Fig.…”

“…The existing co-crystal and cryo-EM structures of CaM with purified full channel and purified fragments containing the A and/or B domains show the A and B domains adopting an ␣-helix secondary structure or assembled in coiled-coil arrangements when embraced by CaM (45)(46)(47)49). However, it is not known whether the free A and B domains of KCNQ channels adopt helical structure in the absence of CaM, despite their common reference as the "A helix" or "B helix."…”

“…Recent structural investigations suggest either that Ca 2ϩ / CaM embraces both the A and B domains of KCNQ1, -4, and -5 and KCNQ2/3 hybrids (45)(46)(47) or that Ca 2ϩ -loading of CaM induces the A domain to be released from the trimeric complex, leaving Ca 2ϩ -loaded CaM to wrap tightly around the B domain alone (48,49). In contrast, a recent solution NMR study of a similar complex of the A and B domains of KCNQ2 and CaM suggested only minor changes in the structure of the complex between low and high [Ca 2ϩ ], arguing against a dramatic structural change in KCNQ channels in response to intracellular rises in [Ca 2ϩ ] (50).…”

Effect of Ca2+on the promiscuous target-protein binding mechanism of calmodulin

“…Both CaM dynamics and motif-dependent target-protein binding have been extensively studied via both experiments [2,8,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and simulations. Indeed, all-atom molecular dynamics (MD) simulations allow for a detailed description with molecular insights.…”