What's the Function of Connexin 32 in the Peripheral Nervous System?

Connexin 32 (Cx32) is a fundamental protein in the peripheral nervous system (PNS) as its mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X), the second most common form of hereditary motor and sensory neuropathy and a demyelinating disease for which there is no effective therapy. Since mutations of the GJB1 gene encoding Cx32 were first reported in 1993, over 450 different mutations associated with CMT1X including missense, frameshift, deletion and non-sense ones have been identified. Despite the availability of a sizable number of studies focusing on normal and mutated Cx32 channel properties, the crucial role played by Cx32 in the PNS has not yet been elucidated, as well as the molecular pathogenesis of CMT1X. Is Cx32 fundamental during a particular phase of Schwann cell (SC) life? Are Cx32 paired (gap junction, GJ) channels in myelinated SCs important for peripheral nerve homeostasis? The attractive hypothesis that short coupling of adjacent myelin layers by Cx32 GJs is required for efficient diffusion of K+ and signaling molecules is still debated, while a growing body of evidence is supporting other possible functions of Cx32 in the PNS, mainly related to Cx32 unpaired channels (hemichannels), which could be involved in a purinergic-dependent pathway controlling myelination. Here we review the intriguing puzzle of findings about Cx32 function and dysfunction, discussing possible directions for future investigation

What’s the Function of Connexin 32 in the Peripheral Nervous System?

Connexin 32 (Cx32) is a fundamental protein in the peripheral nervous system (PNS) as its mutations cause the X-linked form of Charcot–Marie–Tooth disease (CMT1X), the second most common form of hereditary motor and sensory neuropathy and a demyelinating disease for which there is no effective therapy. Since mutations of the GJB1 gene encoding Cx32 were first reported in 1993, over 450 different mutations associated with CMT1X including missense, frameshift, deletion and non-sense ones have been identified. Despite the availability of a sizable number of studies focusing on normal and mutated Cx32 channel properties, the crucial role played by Cx32 in the PNS has not yet been elucidated, as well as the molecular pathogenesis of CMT1X. Is Cx32 fundamental during a particular phase of Schwann cell (SC) life? Are Cx32 paired (gap junction, GJ) channels in myelinated SCs important for peripheral nerve homeostasis? The attractive hypothesis that short coupling of adjacent myelin layers by Cx32 GJs is required for efficient diffusion of K+ and signaling molecules is still debated, while a growing body of evidence is supporting other possible functions of Cx32 in the PNS, mainly related to Cx32 unpaired channels (hemichannels), which could be involved in a purinergic-dependent pathway controlling myelination. Here we review the intriguing puzzle of findings about Cx32 function and dysfunction, discussing possible directions for future investigation

Structure of the connexin-43 gap junction channel in a putative closed state

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state

The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone

BAAV Mediated GJB2 Gene Transfer Restores Gap Junction Coupling in Cochlear Organotypic Cultures from Deaf Cx26Sox10Cre Mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans

Calcium Dynamics in Inner Ear Health and Disease

Ca2+ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. The genetic approach is proving fundamental in unravelling the molecular basis of Ca2+ function in the control of these key cellular processes. Ablation or missense mutations of the PMCA2 Ca2+-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we studied PMCA2 Ca2+-extrusion in hair cells of utricle organotypic cultures from neonatal mice inner ear. Confocal Ca2+ imaging showed that the dissipation of stereociliary Ca2+ transients, induced by cytosolic photoliberation, was compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler and Oblivion mice. Alteration of the intracellular Ca2+ concentration ( ) can trouble the finely tuned control mechanisms of signal transduction, thus resulting as a fundamental physiological parameter to be investigated in the comprehension of deafness mechanisms. By comparing our experimental fluorescence data with those derived from Monte Carlo numerical simulations, we provided a novel method to effectively deconvolve within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. Data analysis performed within this environment indicates that changes of hair cell basolateral during synaptic transmission are primarily controlled by the endogenous Ca2+ buffers at both short (< 1 micron) and long (tens of microns) distances from the presynaptic active zones. Furthermore, we provided quantitative estimates of concentration and kinetics of the endogenous Ca2+-buffers and Ca2+-ATPases in frog vestibular hair cells. We successfully applied mathematical models also in the study of channel permeability to second messengers of gap junctions, intercellular channels connecting supporting cells of the organ of Corti. In particular, it\u2019s known that defective permeation of cAMP or inositol 1,4,5-trisphosphate through gap junction channels is associated with peripheral neuropathies and deafness, respectively. Our model permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways

Defects in the ATP2B2 gene causing hereditary hearing and balance loss in mice and humans: a biophysical study of normal and mutated PMCA2 pump function.

Ca2+ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. The genetic approach is proving fundamental in unravelling the molecular basis of these important biological functions. In particular, ablation or missense mutations of the PMCA2 Ca2+-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we used a combination of confocal fluorescence microscopy and cytosolic Ca2+ photoliberation. The study of Ca2+-extrusion in hair cells from neonatal mice inner ear permitted us to show that Ca2+ extrusion was compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler and Oblivion mice. We suggest that the consequent reduced endolymphatic Ca2+ concentration can trouble the finely tuned control mechanisms of signal transduction, eventually resulting in hair cell death

A biophysical approach to the study of the structure and function of connexin channel nanopores.

Objective: Connexins are transmembrane proteins that form intercellular junctional channels in vertebrates and are known or suspected to be involved in a wide variety of biological processes including cardiac development and function, hearing, haematopoesis, regeneration, lens transparency, fertility, immune system function and protection from oxidative stress. Connexin mutations can cause developmental and physiological defects, and link to various diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP 3 ) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single-gap junction channels to second messengers. Study design : Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fl uorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP 3 . Results: The unitary permeabilities to cAMP (47 \ub1 15 710\u20133 \ub5m3/s) and InsP3 (60 \ub1 12 710\u20133 \ub5m3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 \ub1 3 710\u20133 \ub5m3/s), an exogenous tracer. Conclusion: This method permits quantifi cation of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signalling pathways

PMCA2 pump mutations and hereditary deafness

Hair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K+ and Ca2+ ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter release at the cell basolateral pole. Ca2+ influx into the stereocilia compartment is counteracted by the unusual w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants, increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca2+ concentration ([Ca2+]c). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse models in which the pump is genetically ablated or mutated show hearing and balance impairment, which correlates with defects in homeostatic regulation of stereociliary [Ca2+]c, decreased sensitivity of mechanotransduction channels to hair bundle displacement and progressive degeneration of the organ of Corti. These results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival, and provide mechanistic insight into the etiology of deafness and vestibular disorders

Ca 2+ signaling, apoptosis and autophagy in the developing cochlea: milestones to hearing acquisition

In mammals, the sense of hearing arises through a complex sequence of morphogenetic events that drive the sculpting of the auditory sensory epithelium into its terminally functional three-dimensional shape. While the majority of the underlying mechanisms remain unknown, it has become increasingly clear that Ca2+ signaling is at center stage and plays numerous fundamental roles both in the sensory hair cells and in the matrix of non-sensory, epithelial and supporting cells, which embed them and are tightly interconnected by a dense network of gap junctions formed by connexin 26 (Cx26) and connexin 30 (Cx30) protein subunits. In this review, we discuss the intricate interplay between Ca2+ signaling, connexin expression and function, apoptosis and autophagy in the crucial steps that lead to hearing acquisition