Pathogenetic role of the deafness-related M34T mutation of Cx26

Massimiliano Bicego 2 Martina Beltramello 1 Salvatore Melchionda 0 Massimo Carella 0 Valeria Piazza 1 Leopoldo Zelante 0 Feliksas F. Bukauskas 6 Edoardo Arslan 5 Elona Cama 5 Sergio Pantano 1 3 Roberto Bruzzone 4 Paola D'Andrea 2 Fabio Mammano 1 3 7 0 Servizio di Genetica Medica, IRCCS-Ospedale Casa Sollievo della Sofferenza , San Giovanni Rotondo , Italy 1 Istituto Veneto di Medicina Molecolare (VIMM) , Fondazione per la Ricerca Biomedica Avanzata, 35129 Padova , Italy 2 Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, University of Trieste , 34127 Trieste , Italy 3 Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM) 4 De partement de Neuroscience, Institut Pasteur , 75015 Paris , France 5 Servizio di Audiologia e Foniatria, University of Padova , 35128 Padova , Italy 6 Department of Neuroscience, Albert Einstein College of Medicine , Bronx, NY 10461 , USA 7 Dipartimento di Fisica 'G.Galilei', Universita` di Padova , 35131 Padova , Italy Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca21 waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell - cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment. - INTRODUCTION Connexins comprise a multi-gene family of proteins that form the intercellular channels clustered at gap junctions, through which ions, small metabolites and second messengers are exchanged between connected cells (1). Intercellular channels are formed by two half-channels, or connexons, consisting of six connexin subunits that span the plasma membranes of communicating cells and dock in the intercellular space (2). The specific role of connexin channels in the homeostasis of different organs has been illustrated by the association of mutations in several human connexins with a variety of genetic diseases and by the specific phenotypes revealed by targeted connexin gene deletion in mice (3 5). In the inner ear, three connexin isoforms have been found to be expressed in overlapping patterns (6) and their crucial role in organ physiology has been revealed by their implication in different forms of hereditary deafness (7). Thus, mutations in human Cx26 (HCx26), HCx30 and HCx31 (GJB2, GJB6 and GJB3 genes, respectively) have been linked to both syndromic and non-syndromic forms of hearing loss (reviewed in 8). The arrangement of gap junctions between supporting cells of the organ of Corti appears to be the structural basis for the re-circulation of cochlear K ions that flow from the endolymph, where their concentrations are 150 mM, into hair cells upon sound stimulation (9). In this scheme, K ions flow into the hair cells through mechanically gated channels by the combined effect of the positive endolymphatic potential ( 80 mV) and the negative intracellular potential and depolarize them. K ions exit hair cells reaching the interstitial space of the organ of Corti, where they are partly taken up by cochlear supporting cells (10). The excess K concentration that results from auditory signals or sustained deleterious stimuli, such as ischemia or prolonged exposure to high sound pressure levels (reviewed in 11), is thought to be removed via an intercellular pathway delineated by intercellular channels composed of Cx26 and Cx30 that provide a spatial buffering network similar to that of astrocytic gap junctions in the brain (12). Genetic studies have demonstrated that a specific single-base deletion in Cx26, named 35delG, is the most common mutation worldwide, although its prevalence varies with ethnical groups (8). In addition, several missense mutations responsible for either recessive or dominant forms of the disease have been identified (for an updated list, see Rabionet et al. (2002) http://www.iro.es/cx26deaf.html). The first deafness-related Cx26 mutation, which was detected in a dominant pedigree of non-syndromic hearing loss, was a methionine-to-threonine substitution at position 34 (M34T) (13). The pathogenetic role of M34T was initially supported by the results of its functional expression in Xenopus oocytes, which showed a dominant-negative effect of the M34T mutant (14). Further studies on the family first described by Kelsell et al. (13), however, uncovered the association of dermatological signs in deaf patients and identified another dominant mutation in GJB2 segregating with the disease, casting doubts on the significance of the M34T variant. Subsequently, the same M34T substitution was also reported to be responsible for a recessive form of hearing loss (15), whereas several authors described normal hearing in heterozygous carriers of M34T associated with either 35delG or other Cx26 recessive mutations (16 19) and, therefore, considered it a benign polymorphism. More recent in vitro data have shown that in transfected HeLa cells, the M34T variant disrupts either channel formation or its oligomerization (20,21). In contrast, Thonnissen et al. (22) observed low levels of dye transfer between HeLa cells expressing M34T, providing the first evidence that this mutant could traffic to the cell membrane and form intercellular channels, although with reduced efficiency. More recently, Oshima et al. (23) reported that assembly of M34T in HeLa and Sf9 cells resembles that of wild-type human Cx26 (HCx26wt) and that dye transfer in these cells is close to normal. Finally, electrophysiological studies performed in paired Xenopus oocytes concluded that M34T was capable of forming with Cx32 functional heterotypic channels with abnormal gating properties, which allowed to suggest that the channels are not fully open at rest but are activated when positive transjunctional voltages are applied to the M34T side (24). In addition, co-injection of M34T and HCx26wt in oocytes supported a recessive role for the M34T a (...truncated)