The Crossroad of Ion Channels and Calmodulin in Disease

Calmodulin (CaM) is the principal Ca2+ sensor in eukaryotic cells, orchestrating the activity of hundreds of proteins. Disease causing mutations at any of the three genes that encode identical CaM proteins lead to major cardiac dysfunction, revealing the importance in the regulation of excitability. In turn, some mutations at the CaM binding site of ion channels cause similar diseases. Here we provide a summary of the two sides of the partnership between CaM and ion channels, describing the diversity of consequences of mutations at the complementary CaM binding domains.The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek 2017 bG17 kk-2017/000843M50.17.EK.C6) and the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-66910 and RTI2018-097839) provided financial support for this work. E.N. is supported by a predoctoral grant of the Basque Government

Redox regulation of KV7 channels through EF3 hand of calmodulin

Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.Ministerio de Ciencia e Innovacion PID2021-128286NB-100Wellcome Trust 212302/Z/18/ZMedical Research Centre MR/P015727/1Eusko Jaurlaritza IT1707-22 Ekonomiaren Garapen eta Lehiakortasun Saila, Eusko Jaurlaritza BG2019Ministerio de Ciencia e Innovacion RTI2018-097839-B-100Ministerio de Ciencia e Innovacion RTI2018-101269-B-I00Eusko Jaurlaritza IT1165-19 Ekonomiaren Garapen eta Lehiakortasun Saila,Eusko Jaurlaritza KK-2020/00110Eusko Jaurlaritza PRE_2018-2_0082Eusko Jaurlaritza POS_2021_1_0017Eusko Jaurlaritza PRE_2018-2_012

An Epilepsy-Causing Mutation Leads to Co-Translational Misfolding of the Kv7.2 Channel

BACKGROUND: The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. RESULTS: We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. CONCLUSIONS: Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitroThe Government of the Autonomous Community of the Basque Country (IT1165-19 and KK-2020/00110) and the Spanish Ministry of Science and Innovation (RTI2018-097839-B-100 to A.V. and FIS2016-76617-P to A.B.) and FEDER funds and the US National Institute of Neurological Disorders (NINDS) and Stroke Research Project Grant (R01NS083402 to H.J.C.) provided financial support for this work. E.N. and A.M-M. are supported by predoctoral contracts from the Basque Government administered by University of the Basque Country. C.M. was supported by the Basque Government through a Basque Excellence Research Centre (BERC) grant administered by Fundación Biofisika Bizkaia (FBB). J.U. was partially supported by BERC funds. O.R.B. was supported by the Basque Government through a BERC grant administered by Donostia International Physics Center. J.Z. and H.J.C. was supported by the NINDS Research Project Grant #R01NS083402 (PI: H.J.C.)

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca2+. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using N-15-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca2+ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca2+ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca2+.This work was supported by grants from the Spanish Ministry of Education (BFU2012-39883 and BFU2009-07581), the Spanish Ion Channel Initiative Consolider project (CSD2008-00005), and the Basque Government (SAIOTEK SA-2006/00023 and 304211ENA9). A. Alaimo and C. Malo were partially funded by Fundacion Biofisica Bizkaia. J. Fernandez-Orth held a FPI fellowship from the Spanish Ministry of Science and Innovation (BES-2008-002314). A. Alberdi holds a JAE-predoctoral CSIC fellowship cofinanced with European Social Funds. G. Bernardo-Seisdedos holds a fellowship from the Basque Country Government (BFI-2011-159). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript