View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Archivio della Ricerca - Università di Pisa THE JOURNAL • RESEARCH • www.fasebj.org Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies Paola Imbrici,*,1 Concetta Altamura,* Giulia Maria Camerino,* Giuseppe Felice Mangiatordi,* Elena Conte,* † † ‡ Lorenzo Maggi, Raffaella Brugnoni, Kejla Musaraj,* Roberta Caloiero,* Domenico Alberga, § † Rene` Massimiliano Marsano, Giulia Ricci,{ Gabriele Siciliano,{ Orazio Nicolotti,* Marina Mora, † † Pia Bernasconi, Jean-Francois Desaphy,k Renato Mantegazza, and Diana Conte Camerino* ‡ *Department of Pharmacy, Drug Sciences, Department of Physics “M. Merlin,” Istituto Nazionale di Fisica Nucleare and Centro di Tecnologie Innovative per la Rilevazione e l’Elaborazione del Segnale, §Department of Biology, and kDepartment of Biomedical Sciences and Human † Oncology, University of Bari “Aldo Moro,” Bari, Italy; Division of Neurology, Neuroimmunology and Neuromuscular Diseases Unit, Istituti di Ricovero e Cura a Carattere Scientifico Fondazione Istituto Neurologico “Carlo Besta,” Milan, Italy; and {Department of Clinical and Experimental Medicine, Section of Neurology, University of Pisa, Pisa, Italy ABSTRACT: Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after con- traction caused by loss-of-function mutations in the skeletal muscle ClC-1 channel. We report a novel ClC-1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I-J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch-clamp electrophysiology on T335N and ClC-1 wild-type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC-1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I-J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K+ channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.—Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj,K.,Caloiero,R.,Alberga,D.,Marsano,R.M.,Ricci,G.,Siciliano,G.,Nicolotti,O.,Mora,M.,Bernasconi,P., Desaphy, J.-F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies. FASEB J. 30, 3285–3295 (2016). www.fasebj.org KEY WORDS: chloride channel • gene expression • molecular dynamics • patch-clamp • skeletal muscle Myotonia congenita (MC), in its dominant and recessive age of onset and is more severe. Dominant MC encom- form, is the most common skeletal muscle ion channel- passes a wider range of symptoms, from subclinical to opathy and is characterized by muscle stiffness after moderately severe manifestations (1, 2). MC is caused by voluntary effort (1). Usually, recessive MC has an early loss-of-function mutations in the CLCN1 gene that en- codes the voltage-gated chloride channel, ClC-1, which maintains the large shunting chloride conductance of ABBREVIATIONS: 3D, 3-dimensional; AA, atom acceptor; AD, atom donor; H-bond, hydrogen bond; MC, myotonia congenita; MD, molecular dy- muscle fibers (3). namics; Po, open probability; qPCR, quantitative PCR; TBS, Tris-buffered As for other ion channel diseases, functional studies saline; VMD, visual molecular dynamics; WT, wild-type using heterologous expression systems have been decisive 1 Correspondence: Department of Pharmacy, Drug Sciences, University of Bari to clarify molecular mechanisms underlying myotonia. “Aldo Moro,” Via Orabona 4, 70125 Bari, Italy. E-mail: paola.imbrici@uniba Indeed, in a majority of cases, biophysical characterization This is an Open Access article distributed under the terms of the Creative of expressed ClC-1 mutants satisfactorily demonstrated Commons Attribution 4.0 International (CC BY 4.0) (http://creativecommons. that specific ClC-1 defects were the obvious culprit for org/licenses/by/4.0/) which permits unrestricted use, distribution, and re- production in any medium, provided the original work is properly cited. clinical features of patients (4, 5). In vitro studies proved that 2 recessive mutations must be present, each affecting doi: 10.1096/fj.201500079R This article includes supplemental data. Please visit http://www.fasebj.org to the function of 1 allele, to reduce chloride currents suffi- obtain this information. ciently to produce myotonia (6, 7). In contrast, most ClC-1 0892-6638/16/0030-3285 © The Author(s) 3285 Downloaded from www.fasebj.org by (131.114.70.49) on December 11, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 3285-3295. dominant mutants exert a dominant-negative effect on the MATERIALS AND METHODS associated wild-type (WT) subunit in the heterodimeric channel (8); however, in some cases, results of functional Mutagenesis and expression of hClC-1 WT and studies of expressed mutant channels were inadequate to mutant channels predict a clear correlation between genotype and clinical . symptoms, essential to define the most appropriate The c.1004C A mutation was introduced into the plasmid pRcCMV-hClC-1 that contained the full-length WT hClC-1 treatment (9). For instance, myotonic individuals who cDNA by using the QuikChange site-directed mutagenesis kit carry the same ClC-1 mutation can have markedly var- (Stratagene, Santa Clara, CA, USA), as previously described (5). iable expressivity (10). Some mutations may appear re- The complete coding region of the cDNA was sequenced to ex- cessive or dominant in different families, whereas others clude polymerase errors. tsA201 cells were transiently trans- show incomplete penetrance. Several recessive muta- fected with a mixture of hClC-1 WT or T335N (5 mg) and CD8 m tions show no evidence of defective function in heterol- reporter plasmids (1 g) by using the calcium phosphate pre- ogous systems (5, 11, 12), and some dominant mutations cipitation method. Only cells that were decorated with anti-CD8 antibody–coated microbeads (Dynabeads M450; Thermo Fisher do not induce the expected dominant-negative effect on – Scientific Life Sciences, Waltham, MA, USA) were used for patch- the WT subunit (4 6). These evidences suggest that, as clamp recordings. in the case of other monogenic disorders, environ- mental factors and genetic background of patients may Electrophysiology and data analysis further contribute to variability of MC phenotype (10, 13). Thus, to fulfill the limits of heterologous expres- Standard whole-cell patch-clamp recordings were performed at sion, we recently studied muscle biopsies from pa- room temperature (;20°C) by using an Axopatch 200B amplifier tients with recessive MC to identify possible disease (Axon Instruments, Sunnyvale, CA, USA) (5). We measured the modifiers among genes that are involved in muscle I-V relationship and overall apparent open probability in high- excitability (11), demonstrating that biologic samples chloride (134 mM) intracellular solutions to enhance current from affected individuals can yield valuable informa- amplitude and in more physiologic low internal chloride (4 mM) tion for understanding of the myotonic phenotype. solutions. Composition of the extracellular solution was as fol- lows (in mM): 140 NaCl, 4 KCl, 2 CaCl2,1MgCl2, and 5 HEPES, Muscle biopsies can also help achieve a proper di- pH 7.4 with NaOH. High-chloride pipette solution contained (in agnosis of myotonia (14). mM): 130 CsCl, 2 MgCl2, 5 EGTA, and 10 HEPES, pH 7.4 with Another unsolved issue regarding myotonia, which is CsOH. In this condition, cells were clamped at the holding po- common to a significant number of ion channelopathies, is tential of 0 mV. In the low-chloride internal solution, cesium lack of a specific therapy. Myotonic patients are usually chloride was substituted by cesium glutamate and cells were clamped at the holding potential of –95 mV. Pipettes were pulled administered symptomatic treatment, including mex- ; V iletine, a sodium channel blocker, or acetazolamide, a from borosilicate glass and had 2.5 M resistance. Currents were low-pass filtered at 2 kHz and digitized with sampling rates carbonic anhydrase inhibitor, with limited side effects – of 50 kHz by using the Digidata 1440A AD/DA converter (Axon and high percentages of nonresponders (15 17). Ab- Instruments). In high-chloride solution, 400-ms voltage steps sence of direct ClC-1 opener prompts identification of were applied from 2200 mV to +150 mV in 10-mV intervals, each novel targets and development of new drugs. In this re- followed by a 100-ms voltage step at 2105 mV, where tail cur- spect, analysis of patients’ muscle biopsies, together with rents were measured. In low-chloride solution, currents