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.)

Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding

The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenient functionalization of PEG arms with azide and alkyne groups. The resulting conjugates, with a certain helical character in TFE solutions (CD), showed nanomolar affinity in a fluorescence CaM binding in vitro assay, higher than just the sum of the precursor PEG-peptide affinities, thus validating our design. The approach to these first described examples of Kv7.2 CaMBD-mimetics could pave the way to chimeric conjugates merging helices A and B from different Kv7 subunits. This journal isThis research was supported by Consolider-Ingenio CSD2008-00005 (SICI to AV, RGM, and OM), BFU2012-39092-C02-02 (to RGM) and BFU2012-39883 (to AV). M.A.B. thanks the CSIC for a JAEdoc contract from the program “Junta para la Ampliación de Estudios”, co-financed by the ESF. A.A. was supported by Fundación Biofísica Bizkaia and by a Universidad del País Vasco (UPV/EHU) postdoctoral fellowship. C.M. was co-funded by the Spanish Ministry of Economy and Competitiveness (PTA2012) and by Fundación Biofísica Bizkaia.Peer Reviewe

EF3 mediates CA2+ Gating in KV7.2/CAM complexes

Trabajo presentado en el 64th Annual Meeting of Biophysical Society, celebrado en San Diego (Estados Unidos) entre el 15 y el 19 de febrero de 2020.Kv7.2 (KCNQ2) channel is the principal molecular component of the slow voltage-gated non-inactivating K + M-current, a key controller of the neuronal excitability. To structurally and dynamically describe the association of Kv7.2 C-terminal tetramers with calmodulin (CaM) we used NMR spectroscopy, FRET and HS-AFM. The CaM binding domain forms a ring under the membrane, and is composed by two antiparallel helix, named A and B, embraced by CaM in a C-shaped configuration. We have found that the union of Ca 2+ causes the opening of the helix A/B fork by 18¿ and a torsion of the C-lobe around helix A. Monitoring the conformational changes by FRET we have obtained data that suggest that the union of Ca 2+ to EF-3 is mainly responsible of such conformational change. A marginal effect of ~20% is mediated by EF-4, whereas EF-1 and EF-2 apparently plays no role. The movement of the proximal part of helix A is congruent with the displacement of the S6 helix bundle proposed in models for Kv7.2 opening, suggesting that CaM may affect directly the gating machinery of these channels

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

Calmodulin is critical for folding of the Kv7.2 calcium responsive domain as the nascent peptide exits the ribosome

Trabajo presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Protein co-translational missfolding lead to diseases, such as Alzheimer or Parkinson, but little is known about the role of ancillary proteins. We have demonstrated for the first time that the co-translational folding assistance by calmodulin (CaM) to the KV7.2 channel Calcium Responsive Domain (CRD) is disrupted in epileptogenic encephalopathy.1 The force exerted during the early folding events of the nascent chain can be assessed with single residue resolution. We describe here the force profile of folding of the CRD during translation. We find that CaM, the most important calcium modulator in eukaryotic cells, is required to generate early folding events on this domain at critical places. This investigation provides new insights into how a critical KV7.2 channel domain acquires its final functional conformation during co-translational synthesis.Peer reviewe

Differential Regulation of PI(4,5)P2 Sensitivity of Kv7.2 and Kv7.3 Channels by Calmodulin.

HIGHLIGHTS- Calmodulin-dependent Kv7.2 current density without the need of binding calcium.- Kv7.2 current density increase is accompanied with resistance to PI(4,5)P2 depletion.- Kv7.3 current density is insensitive to calmodulin elevation.- Kv7.3 is more sensitive to PI(4,5)P2 depletion in the presence of calmodulin.- Apo-calmodulin influences PI(4,5)P2 dependence in a subunit specific manner.The identification and understanding of critical factors regulating M-current functional density, whose main components are Kv7.2 and Kv7.3 subunits, has profound pathophysiological impact given the important role of the M-current in neuronal excitability control. We report the increase in current density of Kv7.2 channels by calmodulin (CaM) and by a mutant CaM unable to bind Ca2+ (CaM1234) revealing that this potentiation is calcium independent. Furthermore, after co-expressing a CaM binding protein (CaM sponge) to reduce CaM cellular availability, Kv7.2 current density was reduced. Current inhibition after transient depletion of the essential Kv7 co-factor phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by activating Danio rerio voltage sensitive phosphatase (DrVSP) was blunted by co-expressing CaM1234 or the CaM sponge. In addition, CaM-dependent potentiation was occluded by tonic elevation of PI(4,5)P2 levels by PI(4)P5-kinase (PIP5K) expression. In contrast to the effect on homomeric Kv7.2 channels, CaM1234 failed to potentiate heteromeric Kv7.2/3 or homomeric Kv7.3 channels. Sensitivity to PI(4,5)P2 depletion of Kv7.2/3 channels was increased after expression of CaM1234 or the CaM sponge, while that of homomeric Kv7.3 was unaltered. Altogether, the data reveal that apo-CaM influences PI(4,5)P2 dependence of Kv7.2, Kv7.2/3, and of Kv7.3 channels in a subunit specific manner.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-66910-R). CM. was funded by the Spanish Ministry of Economy and Competitiveness (PTA2012) and co-financed by the BERC program of the Basque Government. MT was an Ikerbasque Visiting Professor funded by the Basque Government, and work in MT laboratories was supported by Telethon (GGP15113).Peer reviewedPeer Reviewe

Ion channel based development of biosensors for discovering improved treatments for amyotrophic lateral sclerosis

Resumen del trabajo presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Calmodulin recognizes more than 300 different targets with very little sequence similarity. Those targets control key events on human physiology with great therapeutically potential [1]. We are working on creating a library of biosensors with the aim of capturing the key molecular rearrangements that take place in different full-length ligands, an connecting those events to easily detectable fluorescent signals amenable of high throughput screening. As a proof of concept, we will present the development of a biosensor that recapitulates the most essential effects of Riluzole, the only drug available for treatment of amyotrophic lateral sclerosis.Peer reviewe

Ion channel based development of biosensors for discovering improved treatments for amyotrophic lateral sclerosis

Trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante (España) del 24 al 27 de mayo de 2022.SK channels, widely expressed in excitable cells, contribute to the after-hyperpolarization following an action potential, mediate the intrinsic excitability of neurons, and control resting potential in lymphocytes and erythrocytes. Ca2+ gates their activity via calmodulin, which is bound at the C-terminus. These channels are targets of riluzole, the only known treatment for amyotrophic lateral sclerosis (ALS), which has limited effectiveness. This drug increases the efficacy of Ca2+ to open the channel. Thus, finding new drugs with improved pharmacology with a similar mode of action is of utmost interest. However, the methodology to assess Ca2+ sensitivity by current electrophysiological methods represents a bottleneck for screening programs. An alternative is creating simplified systems, devoid of most of the protein components, amenable to other techniques. The challenge is to capture the main molecular events that take place during activation yet preserving the same pharmacological profile. The aim of this work is to design biosensors based on the SK4 channel gating that recent cryoEM studies have revealed. Based on these structures, we predicted that upon Ca2+ biding, two regions of the structure should come in close proximity, a movement amenable for detection by FRET. However, this first generation of rationally designed biosensors responded in the opposite direction, with a very small signal to noise ratio. After a fresh new redesign, and several phases of optimization, we have obtained a biosensor template that recapitulates the response to Ca2+ and riluzole observed in the full channel. In addition, we have designed a new library of riluzole analogues oriented by structural bioinformatic approaches. These new biosensors could provide a fast, cheap and effective platform for new therapeutic drug screening