A Structural Characterization of the Kv7.2-Kv7.3 M Channel Proximal C-Terminus/Cam Complex

The Kv7 (KCNQ) family plays major roles in fine-tuning neuronal and cardiomyocyte excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminus (CT) bound constitutively by calmodulin (CaM), responsible for tetramerization, trafficking, and gating properties. The CT contains four helices, dubbed A through D. CaM embraces the proximal CT, comprised of anti-parallel helices A and B. An X-ray structure of Kv7.1-CT/CaM showed that the CaM C-lobe in apo form binds helix A, while Ca2+/N-lobe binds helix B. Concatameric channels demonstrated that CaM binds both helices within the same Kv7.1 subunit. Kv7.2 and Kv7.3 are the predominant members found in neural tissues. Together they form the heterotetrameric M channel, named after the IM current it conducts. IM yields larger current amplitudes compared to currents generated by Kv7.2 homomers. Kv7.3 channel conductance is difficult to distinguish from background level and may not exist as a homomeric channel in vivo. Mutations in Kv7.2 or Kv7.3 cause neonatal epileptic encephalopathies and myokymia.We characterized Kv7.2, Kv7.3 and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by light-scattering and SAXS at various Ca2+ concentrations. We tested the chimeric channel and found it to be functional. We subsequently determined the crystal structure of the Q3A-Q2B/CaM complex at high [Ca2+] to 2.0 A resolution. Our results indicate unique interactions responsible for the inter-subunit pairing of Q3A and Q2B while suggesting that Q3A-Q2B/CaM binding may be similar to that observed for Kv7.1-CT/CaM and unlike the model suggested for Kv7.4-CT/CaM. The Q3A-Q2B/CaM structure can be used to rationalize various Kv7.2 channelopathic mutants. Other implications for M-channel structure-function will be discussed.