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Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability ("leaky") or excessive activity ("hyperactive"). We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations. In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody's applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs. Plain English summary: Connexins (Cxs) are proteins essential for communication between cells, forming gap junction channels (GJCs) that connect neighboring cells and hemichannels (HCs) that link the inside of cells to their surroundings. Mutations in the GJB1 gene, which makes Cx32, cause X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare nerve disorder that currently has no cure. Our research studied the effects of a specially designed antibody, abEC1.1-hIgG1, on both normal and mutated Cx32 HCs using a well-established cell model for studying these channels. We discovered that the antibody significantly reduced the movement of calcium ions (Ca2+), which are vital for nerve health, in both normal and mutated channels. Further analysis showed that the antibody's success might be due to how it binds to certain areas of the channels. Our findings suggest that abEC1.1-hIgG1 could be developed into a treatment for CMTX1 and similar conditions. Moreover, this research could help in creating even more effective antibodies for targeting faulty hemichannels. [ABSTRACT FROM AUTHOR]

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues. [ABSTRACT FROM AUTHOR]

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2 loxP/loxP; ROSA26 CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker's organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker's organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function. [ABSTRACT FROM AUTHOR]

GJB2 and GJB6 are adjacent genes encoding connexin 26 (Cx26) and connexin 30 (Cx30), respectively, with overlapping expressions in the inner ear. Both genes are associated with the commonest monogenic hearing disorder, recessive isolated deafness DFNB1. Cx26 plays an important role in auditory development, while the role of Cx30 in hearing remains controversial. Previous studies found that Cx30 knockout mice had severe hearing loss along with a 90% reduction in Cx26, while another Cx30 knockout mouse model showed normal hearing with nearly half of Cx26 preserved. In this study, we used CRISPR/Cas9 technology to establish a new Cx30 knockout mouse model (Cx30−/−), which preserves approximately 70% of Cx26. We found that the 1, 3, and 6-month-old Cx30−/− mice showed mild hearing loss at full frequency. Immunofluorescence and HE staining suggested no significant differences in microstructure of the cochlea between Cx30−/− mice and wild-type mice. However, transmission electron microscopy showed slight cavity-like damage in the stria vascularis of Cx30−/− mice. And Cx30 deficiency reduced the production of endocochlear potential (EP) and the release of ATP, which may have induced hearing loss. Taken together, this study showed that lack of Cx30 can lead to hearing loss with an approximately 30% reduction of Cx26 in the present Cx30 knockout model. Hence, Cx30 may play an important rather than redundant role in hearing development. [ABSTRACT FROM AUTHOR]

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration (${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ oscillations. Graphical Abstract [ABSTRACT FROM AUTHOR]

Autophagy and apoptosis have a complex interplay in the early embryo development. The development of spiral ganglion neurons (SGNs) in addition to Corti's organ in the mammalian cochlea remains crucial in the first two-week postnatal period. To investigate the roles of apoptosis and autophagy in the development of SGNs, light microscopy was used to observe the morphological changes of SGNs. The number of SGNs was decreased from P1 to P7 and plateaued from P10 to P14. Immunohistochemistry results revealed positive expression of cleaved-caspase3, bcl-2, microtubule-associated protein light chain 3-II (LC3-II), Beclin1, and sequestosome 1 (SQSTM1/P62) in SGNs. The apoptotic bodies and autophagosomes and autolysosomes were also identified by transmission electron microscopy at P1 and P7. Real-time PCR and western blotting results revealed that the apoptotic activity peaked at P7 and the autophagy activity was gradually upregulated along with the development. Taken together, our results for the first time showed that autophagy and apoptosis in SGNs play distinct roles during specific developmental phases in a time-dependent manner. [ABSTRACT FROM AUTHOR]

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6−/− COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1−/− with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin–luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods. [ABSTRACT FROM AUTHOR]

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies. [ABSTRACT FROM AUTHOR]

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family. [ABSTRACT FROM AUTHOR]

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1-/- mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1-/-), hemizygous (Panx1+/-) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1-/- mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1-/- cochleae. Hearing sensitivity, outer hair cell-based "cochlear amplifier" and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/- and Panx1-/- mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function. [ABSTRACT FROM AUTHOR]

Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. [ABSTRACT FROM AUTHOR]

Introduction: Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability. Methods: Here, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity. We develop and validate a microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca dynamics, and insulin release during perfusion under 3 or 11 mM glucose. Results: We found that simvastatin reversibly inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (<60 s), occurs without affecting Ca concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours. Conclusions: Our data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale. Funding: University of Padova, EASD Foundation. [ABSTRACT FROM AUTHOR]

Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 μm·s-1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems. [ABSTRACT FROM AUTHOR]

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca2+ homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca2+-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca2+ load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca2+ overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca2+ signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca2+ signal-dependent apoptosis. [ABSTRACT FROM AUTHOR]

Mutations of the GJB2 gene encoding the connexin 26 (Cx26) gap junction protein, which is widely expressed in the inner ear, are the primary cause of hereditary non-syndromic hearing loss in several populations. The deafness-associated single amino acid substitution of methionine 34 (M34) in the first transmembrane helix (TM1) with a threonine (T) ensues in the production of mutant Cx26M34T channels that are correctly synthesized and assembled in the plasma membrane. However, mutant channels overexpressed in HeLa cells retain only 11% of the wild type unitary conductance. Here we extend and rationalize those findings by comparing wild type Cx26 (Cx26WT) and Cx26M34T mutant channels in silico, using molecular dynamics simulations. Our results indicate that the quaternary structure of the Cx26M34T hemichannel is altered at the level of the pore funnel due to the disruption of the hydrophobic interaction between M34 and tryptophan 3 (W3) in the N-terminal helix (NTH). Our simulations also show that external force stimuli applied to the NTHs can detach them from the inner wall of the pore more readily in the mutant than in the wild type hemichannel. These structural alterations significantly increase the free energy barrier encountered by permeating ions, correspondingly decreasing the unitary conductance of the Cx26M34T hemichannel. Our results accord with the proposal that the mutant resides most of the time in a low conductance state. However, the small displacement of the NTHs in our Cx26M34T hemichannel model is not compatible with the formation of a pore plug as in the related Cx26M34A mutant. [ABSTRACT FROM AUTHOR]

Background A variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho-physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity. Results Here, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large-scale optical recordings and a digital phase-sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensors of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that a suitably encoded Vf2.1.Cl signal remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non-sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock-in mice, and by 91% in cultures from connexin30-/- mice, which are profoundly deaf. Conclusions Our methodology allows greater sensitivity (defined as the minimum magnitude of input signal required to produce a specified output signal having a specified signal-to-noise ratio) and better time resolution compared to classical tracer-based techniques. It permitted us to dynamically visualize intercellular connectivity down to the 10th order in non-sensory cell networks of the developing cochlea. We believe that our approach is of general interest and can be seamlessly extended to a variety of biological systems, as well as to other connexin-related disease conditions. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol phosphate kinase type 1γ (PIPKIγ) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI (4,5)P2] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKIγ+/- siblings breed normally and have no reported phenotype. Here we show that adult PIPKIγ+/- mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca2+ signaling activity in cochlear nonsensory cells. Because Ca2+ signaling under these conditions depends on inositol-1,4,5-trisphos-phate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P2, we conclude that (i) PIPKIγ is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P2 pool in the cell syncytia that supports auditory hair cells; (ii) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKIγ+/- mice. This mouse model also suggests that PIPKIγ may determine the level of gap junction contribution to cochlear development. [ABSTRACT FROM AUTHOR]

The permeability of gap junction channels to metabolites, and not simply to small inorganic ions, is likely to play an important role in development, physiology as well as in etiology of several diseases. Here, we combined dual patch clamp and fluorescence imaging techniques with molecular dynamics (MD) simulations to investigate the permeation of calcein, a relatively large fluorescent tracer (MW 622 Da) through homomeric gap junction channels formed by wild type human connexin26 (hCx26wt) protomers. Our experimental data indicate that the unitary flux of calcein driven by a 125 µM concentration difference is Jpore = 226 molecule/s per channel. In the light of Eyring transition state theory adapted for the liquid phase, this value corresponds to an energy barrier of ~20 kBT (where kB is the Boltzmann constant and T is absolute temperature). The barrier predicted by our MD simulations, based on the 3.5 Å X-ray structural model of the hCx26wt gap junction channel, is ~45 kBT. The main contributions to the energetics of calcein permeation originated from the interaction between the permeating molecule and the charged aminoacids lining the channel pore. Assigning a fake zero total charge to the calcein molecule yielded a value for the barrier height compatible with the experimental data. These results can be accounted for by two different (although not mutually exclusive) hypotheses: (1) the X-ray model of the hCx26wt gap junction channel is not representative of a fully open state; (2) post translational modifications affecting the hCx26wt protein in our expression system differed from the modifications undergone by the proteins in the conditions used to obtain the crystal structure. Hypothesis (1) is compatible with data indicating that, only 10% or less of the channels forming a gap junction plaque are in the open state, and therefore the averaging procedure intrinsic in the generation of the crystal structure data more closely reflects that of a closed channel. Hypothesis (2) is compatible with recent mass spectrometry data and implies that the charge of several amino acid side chains may have been altered, thus modifying substantially the permeation properties of the channels in living cells. [ABSTRACT FROM AUTHOR]

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 (InsP3) 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 fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP3. Results: The unitary permeabilities to cAMP (47 ± 15 × 10–3 μm3/s) and InsP3 (60 ± 12 × 10–3 μm3/s) were similar, but substantially larger than the unitary permeability to Lucifer Yellow (LY; 7 ± 3 × 10–3 μm3/s), an exogenous tracer. Conclusion: This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signalling pathways. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two-photon and optical coherence tomography (OCT) based on GRIN optical probes permit in-vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low-order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality. [ABSTRACT FROM AUTHOR]

Ca2+ acts as a fundamental signal transduction element in inner ear, delivering information about sound, acceleration and gravity through a small number of mechanotransduction channels in the hair cell stereocilia and voltage activated Ca2+ channels at the ribbon synapse, where it drives neurotransmission. The mechanotransduction process relies on the endocochlear potential, an electrical potential difference between endolymph and perilymph, the two fluids bathing respectively the apical and basolateral membrane of the cells in the organ of Corti. In mouse models, deafness and lack or reduction of the endocochlear potential correlate with ablation of connexin (Cx) 26 or 30. These Cxs form heteromeric channels assembled in a network of gap junction plaques connecting the supporting and epithelial cells of the organ of Corti presumably for K+ recycle and transfer of key metabolites, for example, the Ca2+‐mobilizing second messenger IP3. Ca2+ signaling in these cells could play a crucial role in regulating Cx expression and function. Another district where Ca2+ signaling alterations link to hearing loss is hair cell apex, where ablation or missense mutations of the PMCA2 Ca2+‐pump of the stereocilia cause deafness and loss of balance. If less Ca2+ is exported from the stereocilia, as in the PMCA2 mouse mutants, Ca2+ concentration in endolymph is expected to fall causing an alteration of the mechanotransduction process. This may provide a clue as to why, in some cases, PMCA2 mutations potentiated the deafness phenotype induced by coexisting mutations of cadherin‐23 (Usher syndrome type 1D), a single pass membrane Ca2+ binding protein that is abundantly expressed in the stereocilia. [ABSTRACT FROM AUTHOR]

The genes GJB2 and GJB6 respectively encoding transmembrane proteins connexin 26 (C×26) and connexin 30 (C×30) are found within 50 kb of each other in the DFNB1 complex deafness locus on chromosome 13ql2. Up to 50% of all patients with autosomal recessive non-syndromic prelingual deafness in different populations present with mutations or deletions in this locus. C×26 and C×30 are abundantly expressed in the inner ear and, in recent years, have been shown to form hemichannels that release ATP from the endolymphatic surface of supporting and epithelial cells of the organ of Corti (OoC), as well as gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, ensuing in the regenerative propagation of intercellular Ca2+ signals (ICS) across these coupled cells. The range of ICS propagation in the OoC is sensitive to the concentration of extracellular divalent cations and to ectonucleotidase activity. Strictly related oscillations of the intracellular free Ca2+ concentration ([Ca2+]i) in cochlear supporting cells are also evoked by nanomolar concentrations of ATP on the endolymphatic surface of the OoC. ICS and [Ca2+]i oscillations are of great interest in relation to the responses evoked by damaging stimuli delivered to hair cells, and may play a crucial role in development of the OoC and the acquisition of hearing. [ABSTRACT FROM AUTHOR]

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, instigating the regenerative propagation of intercellular Ca2+ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP3) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP3 to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca2+ concentration ([Ca2+]i) coordinated by the propagation of Ca2+ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP3 or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. [ABSTRACT FROM AUTHOR]

Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes (GJB2 and GJB6, respectively) that are found within 50 kb in the same complex deafness locus, DFNB1. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. To explore connexin coregulation, we manipulated gene expression using the bovine adeno-associated virus. Over-expression of Cx30 in the Cx30 KO mouse by transduction with bovine adeno-associated virus restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca[sup2+] signals. Ablation of Cx26 by transduction of Cx26[suplox/loxP] cultures with a Cre recombinase vector caused concurrent downregulation of Cx30 and impaired intercellular communication. The coordinated regulation of Cx26 and Cx30 expression appears to occur as a result of signaling through PLC and the NF-κB pathway, because activation of IP[sub3]-mediated Ca[sup2+] responses by stimulation of P2Y receptors for 20 mm with 20 nM ATP increased the levels of Cx26 transcripts in Cx30 KO cultures. This effect was inhibited by expressing a stable form of the IκB repressor protein that prevents activation/translocation of NF-κB. Thus, our data reveal a Ca[sup2+]-dependent control in the expression of inner ear connexins implicated in hereditary deafness as well as insight into the hitherto unexplained observation that some deafness-associated DFNB1 alleles are characterized by hereditable reduction of both GJB2 and GJB6 expression. [ABSTRACT FROM AUTHOR]

Extracellular ATP controls various signaling systems including propagation of intercellular Ca[sup2+] signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP[sub3]-generating agonist and evokes Ca[sup2+] responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP[sub3] elicited Ca[sup2+] responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca[sup2+] signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WI cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Pxl), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca[sup2+] responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca[sup2+] responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca[sup2+] signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca[sup2+]-mobilizing second messengers across coupled cells. [ABSTRACT FROM AUTHOR]

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 semidominant 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. [ABSTRACT FROM AUTHOR]

Connexins are plasma membrane proteins that associate in hexameric complexes to form channels named connexons. Two connexons in neighboring cells may dock to form a ''gap junction'' channel, i.e. an intercellular conduit that permits the direct exchange of solutes between the cytoplasm of adjacent cells and thus mediate cell-cell ion and metabolic signaling. The lack of high resolution data for connexon structures has hampered so far the study of the structure-function relationships that link molecular effects of disease-causing mutations with their observed phenotypes. Here we present a combination of modeling techniques and molecular dynamics (MD) to infer side chain positions starting from low resolution structures containing only Cα atoms. We validated this procedure on the structure of the KcsA potassium channel, which is solved at atomic resolution. We then produced a fully atomistic model of a homotypic Cx32 connexon starting from a published model of the Cα carbons arrangement for the connexin transmembrane helices, to which we added extracellular and cytoplasmic loops. To achieve structural relaxation within a realistic environment, we used MD simulations inserted in an explicit solvent-membrane context and we subsequently checked predictions of putative side chain positions and interactions in the Cx32 connexon against a vast body of experimental reports. Our results provide new mechanistic insights into the effects of numerous spontaneous mutations and their implication in connexin-related pathologies. This model constitutes a step forward towards a structurally detailed description of the gap junction architecture and provides a structural platform to plan new biochemical and biophysical experiments aimed at elucidating the structure of connexin channels and hemichannels. [ABSTRACT FROM AUTHOR]

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) 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. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP3. The unitary permeabilities to cAMP (47 × 10−3 ± 15 × 10−3 μm3/s) and InsP3 (60 × 10−3 ± 12 × 10−3 μm3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 ± 3 × 10−3 μm3/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways. [ABSTRACT FROM AUTHOR]

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca2+-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P3 into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca2+ throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P3 to an extent sufficient to impair the propagation of Ca2+ waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease. [ABSTRACT FROM AUTHOR]

Sounds originating from within the inner ear, known as otoacoustic emissions (OAEs), are widely exploited in clinical practice but the mechanisms underlying their generation are not entirely clear. Here we present simulation results and theoretical considerations based on a hydrodynamic model of the human inner ear. Simulations show that, if the cochlear amplifier (CA) gain is a smooth function of position within the active cochlea, filtering performed by a middle ear with an irregular, i.e., nonsmooth, forward transfer function suffices to produce irregular and long-lasting residual oscillations of cochlear basilar membrane (BM) at selected frequencies. Feeding back to the middle ear through hydrodynamic coupling afforded by the cochlear fluid, these oscillations are detected as transient evoked OAEs in the ear canal. If, in addition, the CA gain profile is affected by irregularities, residual BM oscillations are even more irregular and tend to evolve towards self-sustaining oscillations at the loci of gain irregularities. Correspondingly, the spectrum of transient evoked OAEs exhibits sharp peaks. If both the CA gain and the middle-ear forward transfer function are smooth, residual BM oscillations have regular waveforms and extinguish rapidly. In this case no emissions are produced. Finally, and paradoxically albeit consistent with observations, simulating localized damage to the CA results in self-sustaining BM oscillations at the characteristic frequencies (CFs) of the sites adjacent to the damage region, accompanied by generation of spontaneous OAEs. Under these conditions, stimulus-frequency OAEs, with typical modulation patterns, are also observed for inputs near hearing threshold. This approach can be exploited to provide novel diagnostic tools and a better understanding of key phenomena relevant for hearing science. [ABSTRACT FROM AUTHOR]

Investigates the role played by intracellular calcium stores in afferent transmission at the presynaptic level. Function of hair cells; Process involved in the calcium-induced calcium release; Manner in which the rare of transmitter release at the synapse between the hair cell and the afferent terminal is modulated.

Simple Summary: Bystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and may greatly influence therapy outcome. Although the limited data available suggest a role for intercellular gap junction channels, far less is known about the role of connexin hemichannels. Here, we investigated bystander effects induced by photodynamic therapy in syngeneic murine melanoma models in vivo. We determined that (i) photoactivation of a photosensitizer triggered calcium-dependent cell death pathways in both irradiated and bystander tumor cells; (ii) hemichannel activity and adenosine triphosphate release were key factors for the induction of bystander cell death; and (iii) bystander cell killing and antitumor response elicited by photodynamic therapy were greatly enhanced by combination treatment with S-nitrosoglutathione, which promoted hemichannel opening in these experimental conditions. Therefore, these findings in a preclinical model have important translational potential. In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT. [ABSTRACT FROM AUTHOR]

Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, −76±4 mV ( n=19) and input conductance (10±3 nS at −70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K currents in the cell were identified. One current, activated positive to −100 mV, was reduced by 5 mM BaCl. The other current, activated above −40 mV, was reduced by 100 μM 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier. [ABSTRACT FROM AUTHOR]

Reports on the distortion of the cochlea partition under the action of electrically driven hair cell length changes. Production of place-specific vibration of a basilar membrane; Schematic drawing of the cochlea partition cross-section; Reduction of partition motion by salicylate.

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called "bystander effects". To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2−∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2−∙ production in these bystander cells. Our results support a key role for ER–mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing. [ABSTRACT FROM AUTHOR]

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine‐tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non‐sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non‐sensory cells of the greater epithelial ridge cause, via ATP‐induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience‐independent Ca2+ signals from sensory and non‐sensory cells. Synopsis: How Ca2+‐dependent activities guide the early development of specialized outer hair cells and their afferent neuronal connectivity is poorly understood. Spontaneous Ca2+ waves in non‐sensory cells increase and synchronize Ca2+ signalling in outer hair cells are now found to promote functional differentiation and innervation of sensory cells. Immature outer hair cells (OHCs) spontaneously show transient Ca2+ signals at around birth in mice.Calcium waves in non‐sensory cells are able to increase and synchronise Ca2+ signals in OHCs.The extracellular modulation of OHC Ca2+ signalling activity requires ATP and activation of P2X3 receptors in OHCs.Loss of Ca2+ waves in cochlear non‐sensory cells prevents the acquisition of the normal innervation pattern of OHCs. [ABSTRACT FROM AUTHOR]