BackgroundCisplatin, an effective chemotherapy, is a cause of ototoxicity. It enters inner ear cells through Organic Cation Transporter 2 (OCT2). Recently, we identified another cochlear transporter, Multidrug and Toxin Extrusion Protein 1 (MATE1); it allows cisplatin efflux in renal cells. OCT2 and MATE1 appear to work together in eliminating renal toxins. 4-aminopyridine (4-AP), a known blocker of voltage-dependent K+ channels, blocks OCT2 but not MATE1 in the kidney. We hypothesize that 4-AP could prevent cisplatin from entering cochlear cells, without inhibiting the efflux of cisplatin that may have already entered. This could enhance clearance, mitigating ototoxicity.ObjectiveDetermine the otoprotective potential of 4-AP in vitro following cisplatin exposure.MethodsMice cochlear explants were treated with cisplatin (20 μM), with or without 4-AP (500 μM) for 24 h, evaluated for caspase 3/7 activity with Cell Event®, visualized with confocal and phase contrast microscopy. Moreover, HeLa cells and cochlear explants were exposed to cisplatin (75 μM and 30 μM, respectively) with or without 4-AP in the explants and were evaluated for cisplatin-DNA adducts and cleaved caspase-3 immunohistochemistry.ResultsFor cochlear explants exposed to cisplatin, Cell Event® staining occurred in the supporting cells, medial to the hair cells, and in the organ of Corti. No significant differences were found between cisplatin-exposed explants with or without 4-AP. In HeLa cells, consistent cellular staining for cisplatin-DNA adducts and cleaved caspase-3 was observed. However, this was not seen in cochlear explants, and there was no significant decrease in staining levels with 4-AP co-treatment.ConclusionsIn this in vitro study, no otoprotective potential of 4-AP against cisplatin-induced ototoxicity was observed. While 4-AP blocks OCT2, it did not decrease cisplatin-DNA adducts in cochlear cells. Further research into OCT2 and MATE1 transport mechanisms is needed.

Connexins (Cxs) function as gap junction (GJ) channels and hemichannels that mediate intercellular and transmembrane signaling, respectively. Here, we investigated the proximal segment of the first extracellular loop, E1, of two closely related Cxs, Cx26 and Cx30, that share widespread expression in the cochlea. Computational studies of Cx26 proposed that this segment of E1 contains a parahelix and functions in gating. The sequence of the parahelix is identical between Cx26 and Cx30 except for an Ala/Glu difference at position 49. We show through cysteine-scanning and mutational analyses that position 49 is pore-lining and interacts with the adjacent Asp50 residue to impact hemichannel functionality. When both positions 49 and 50 are charged, as occurs naturally in Cx30, the hemichannel function is dampened. Co-expression of Cx30 with Cx26(D50N), the most common mutation associated with keratitis-ichthyosis-deafness syndrome, results in robust hemichannel currents indicating that position 49-50 interactions are relevant in heteromerically assembled hemichannels. Cysteine substitution at position 49 in either Cx26 or Cx30 results in tonic inhibition of hemichannels, both through disulfide formation and high-affinity metal coordination, suggestive of a flexible region of the pore that can narrow substantially. These effects are absent in GJ channels, which exhibit wild-type functionality. Examination of postnatal cochlear explants suggests that Cx30 expression is associated with reduced propagation of Ca2+ waves. Overall, these data identify a pore locus in E1 of Cx26 and Cx30 that impacts hemichannel functionality and provide new considerations for understanding the roles of these connexins in cochlear function.

BackgroundCisplatin appears to enter the cochlear cells through the organic cation transporter 2 (OCT2). There is recent evidence that multidrug and toxin extrusion protein 1 (MATE1) is involved in cisplatin-induced nephrotoxicity. Its presence and role in the ear are unknown.Aims/ObjectivesEvaluate the presence and localization of MATE1, and determine the localization of OCT2, in the cochlea. Evaluate cisplatin uptake with regard to MATE1 and OCT2 expression.Material and MethodsMurine cochlear explants and paraffin-embedded cochleae were evaluated with immunohistochemistry for OCT2 and MATE1. Explant cultures were also treated with Texas Red cisplatin to determine their cellular uptake.ResultsMATE1 is present in the cochlea. Most intense labeling of MATE1 and OCT2 was seen in the outer hair cells (OHCs) and pillar cells, respectively. Both transporters were observed in the spiral ganglion neurons and stria vascularis. Expression levels of OCT2 and MATE1 decreased following cisplatin exposure. Texas Red cisplatin staining was strong in OHCs and pillar cells.Conclusions and SignificanceTo the best of our knowledge, this is the first study demonstrating the presence and localization of MATE1 in the cochlea. OCT2 labeling was seen in pillar cells. Consistently, OHCs and pillar cells uptake Texas Red cisplatin.

Some mutations in gap junction protein Connexin 26 (Cx26) lead to syndromic deafness, where hearing impairment is associated with skin disease, like in Keratitis Ichthyosis Deafness (KID) syndrome. This condition has been linked to hyperactivity of connexin hemichannels but this has never been demonstrated in cochlear tissue. Moreover, some KID mutants, like Cx26S17F, form hyperactive HCs only when co-expressed with other wild-type connexins. In this work, we evaluated the functional consequences of expressing a KID syndromic mutation, Cx26S17F, in the transgenic mouse cochlea and whether co-expression of Cx26S17F and Cx30 leads to the formation of hyperactive HCs. Indeed, we found that cochlear explants from a constitutive knock-in Cx26S17F mouse or conditional in vitro cochlear expression of Cx26S17F produces hyperactive HCs in supporting cells of the organ of Corti. These conditions also produce loss of hair cells stereocilia. In supporting cells, we found high co-localization between Cx26S17F and Cx30. The functional properties of HCs formed in cells co-expressing Cx26S17F and Cx30 were also studied in oocytes and HeLa cells. Under the recording conditions used in this study Cx26S17F did not form functional HCs and GJCs, but cells co-expressing Cx26S17F and Cx30 present hyperactive HCs insensitive to HCs blockers, Ca2+ and La3+, resulting in more Ca2+ influx and cellular damage. Molecular dynamic analysis of putative heteromeric HC formed by Cx26S17F and Cx30 presents alterations in extracellular Ca2+ binding sites. These results support that in KID syndrome, hyperactive HCs are formed by the interaction between Cx26S17F and Cx30 in supporting cells probably causing damage to hair cells associated to deafness.

Cisplatin is a known ototoxic chemotherapy drug, causing irreversible hearing loss. Evidence has shown that cisplatin causes inner ear damage as a result of adduct formation, a proinflammatory environment and the generation of reactive oxygen species within the inner ear. The main cochlear targets for cisplatin are commonly known to be the outer hair cells, the stria vascularis and the spiral ganglion neurons. Further evidence has shown that certain transporters can mediate cisplatin influx into the inner ear cells including organic cation transporter 2 (OCT2) and the copper transporter Ctr1. However, the expression profiles for these transporters within inner ear cells are not consistent in the literature, and expression of OCT2 and Ctr1 has also been observed in supporting cells. Organ of Corti supporting cells are essential for hair cell activity and survival. Special interest has been devoted to gap junction expression by these cells as certain mutations have been linked to hearing loss. Interestingly, cisplatin appears to affect connexin expression in the inner ear. While investigations regarding cisplatin-induced hearing loss have been focused mainly on the known targets previously mentioned, the role of supporting cells for cisplatin-induced ototoxicity has been overlooked. In this mini review, we discuss the implications of supporting cells expressing OCT2 and Ctr1 as well as the potential role of gap junctions in cisplatin-induced cytotoxicity.

Background. Evidence-based information on genetic sensorineural hearing loss in Latin America is limited, hindering the advancement of related clinical practice and the development of relevant healthcare policies in the field. This study describes sociodemographic and clinical characteristics of a group of Chilean participants with congenital, childhood, progressive or late-onset sensorineural hearing loss of unknown etiology; all non-genetic causes of hearing loss were excluded. Methods. A quantitative study, with a non-experimental, observational, cross-sectional design and a descriptive scope. From 978 patients diagnosed with SNHL, 286 subjects fulfilled the inclusion criteria, and 138 patients accepted to participate. Results. The most frequent audiological-profile was symmetrical-bilateral-profound-SNHL with a sloping pattern. The median age at audiological diagnosis was 24.5 months (IQR:12-53) and at first-time Hearing-Assistive-Device-use (HAD-use) was 30 months (IQR:13-69). 71% of HAD were financed by public resources. Conclusion. The age at audiological diagnosis in SNHL of suspected genetic causes is still far from international standards. These results are valuable for public health research and policy development, not only for the Chilean population, but also for other Hispanic communities and other middle-high income countries.

Diabetic retinopathy (DR) is one of the main causes of vision loss in the working age population. It is characterized by a progressive deterioration of the retinal microvasculature, caused by long-term metabolic alterations inherent to diabetes, leading to a progressive loss of retinal integrity and function. The mammalian retina presents an orderly layered structure that executes initial but complex visual processing and analysis. Gap junction channels (GJC) forming electrical synapses are present in each retinal layer and contribute to the communication between different cell types. In addition, connexin hemichannels (HCs) have emerged as relevant players that influence diverse physiological and pathological processes in the retina. This article highlights the impact of diabetic conditions on GJC and HCs physiology and their involvement in DR pathogenesis. Microvascular damage and concomitant loss of endothelial cells and pericytes are related to alterations in gap junction intercellular communication (GJIC) and decreased connexin 43 (Cx43) expression. On the other hand, it has been shown that the expression and activity of HCs are upregulated in DR, becoming a key element in the establishment of proinflammatory conditions that emerge during hyperglycemia. Hence, novel connexin HCs blockers or drugs to enhance GJIC are promising tools for the development of pharmacological interventions for diabetic retinopathy, and initial in vitro and in vivo studies have shown favorable results in this regard.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels.

Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca2+ show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.

Mutation of the GJB2 gene, which encodes the connexin 26 (Cx26) gap junction (GJ) protein, is the most common cause of hereditary, sensorineural hearing loss. Cx26 is not expressed in hair cells, but is widely expressed throughout the non-sensory epithelial cells of the cochlea. Most GJB2 mutations produce non-syndromic deafness, but a subset produces syndromic deafness in which profound hearing loss is accompanied by a diverse array of infectious and neoplastic cutaneous disorders that can be fatal. Although GJ channels, which are assembled by the docking of two, so-called hemichannels (HCs), have been the main focus of deafness-associated disease models, it is now evident that the HCs themselves can function in the absence of docking and contribute to signaling across the cell membrane as a novel class of ion channel. A notable feature of syndromic deafness mutants is that the HCs exhibit aberrant behaviors providing a plausible basis for disease that is associated with excessive or altered contributions of Cx26 HCs that, in turn, lead to compromised cell integrity. Here we discuss some of the aberrant Cx26 HC properties that have been described for mutants associated with keratitis-ichthyosis-deafness (KID) syndrome, a particularly severe Cx26-associated syndrome, which shed light on genotype-phenotype relationships and causes underlying cochlear dysfunction.

Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca2+. This study presents two new possible contributing factors, pH and Zn2+. Plasma pH levels and micromolar concentrations of Zn2+ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H+ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn2+. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn2+ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn2+. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.

Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extracellular Ca(2+), decreased unitary conductance, increased open hemichannel current rectification and voltage-shifted activation. We demonstrate that D50 is a pore-lining residue and that negative charge at this position strongly influences open hemichannel properties. Examination of two putative intersubunit interactions involving D50 suggested by the Cx26 crystal structure, K61-D50 and Q48-D50, showed no evidence of a K61-D50 interaction in hemichannels. However, our data suggest that Q48 and D50 interact and disruption of this interaction shifts hemichannel activation positive along the voltage axis. Additional shifts in activation by extracellular Ca(2+) remained in the absence of a D50-Q48 interaction but required an Asp or Glu at position 50, suggesting a separate electrostatic mechanism that critically involves this position. In gap junction (GJ) channels, D50 substitutions produced loss of function, whereas K61 substitutions functioned as GJ channels but not as hemichannels. These data demonstrate that D50 exerts effects on Cx26 hemichannel and GJ channel function as a result of its dual role as a pore residue and a component of an intersubunit complex in the extracellular region of the hemichannel. Differences in the effects of substitutions in GJ channels and hemichannels suggest that perturbations in structure occur upon hemichannel docking that significantly impact function. Collectively, these data provide insight into Cx26 structure-function and the underlying bases for the phenotypes associated with KID syndrome patients carrying the D50N mutation.

The synchronization of numerous cellular events requires complex electric and metabolic cell-cell interactions. Connexins are a family of membrane proteins that constitute the molecular basis of two kinds of channels: gap junction channels (GJCs), which allow direct cytoplasm-cytoplasm communication, and hemichannels (HCs) that provide a pathway for exchanges between the intra and extra-cellular milieu. Both kind of connexin-based channels support intercellular communication via intercellular propagation of calcium waves. Here, we review evidence supporting the role of Ca2+ in the regulation of GJCs and HCs formed by connexins. Also it is speculated how these connexin-based channels could contribute to the propagation of intercellular Ca2+ signals.

The anti-malarial drug quinine and its quaternary derivative N-benzylquininium (BQ+) have been shown to inhibit gap junction (GJ) channels with specificity for Cx50 over its closely related homologue Cx46. Here, we examined the mechanism of BQ+ action using undocked Cx46 and Cx50 hemichannels, which are more amenable to analyses at the single-channel level. We found that BQ+ (300 µM–1 mM) robustly inhibited Cx50, but not Cx46, hemichannel currents, indicating that the Cx selectivity of BQ+ is preserved in both hemichannel and GJ channel configurations. BQ+ reduced Cx50 hemichannel open probability (Po) without appreciably altering unitary conductance of the fully open state and was effective when added from either extracellular or cytoplasmic sides. The reductions in Po were dependent on BQ+ concentration with a Hill coefficient of 1.8, suggesting binding of at least two BQ+ molecules. Inhibition by BQ+ was voltage dependent, promoted by hyperpolarization from the extracellular side and conversely by depolarization from the cytoplasmic side. These results are consistent with binding of BQ+ in the pore. Substitution of the N-terminal (NT) domain of Cx46 into Cx50 significantly impaired inhibition by BQ+. The NT domain contributes to the formation of the wide cytoplasmic vestibule of the pore and, thus, may contribute to the binding of BQ+. Single-channel analyses showed that BQ+ induced transitions that did not resemble pore block, but rather transitions indistinguishable from the intrinsic gating events ascribed to loop gating, one of two mechanisms that gate Cx channels. Moreover, BQ+ decreased mean open time and increased mean closed time, indicating that inhibition consists of an increase in hemichannel closing rate as well as a stabilization of the closed state. Collectively, these data suggest a mechanism of action for BQ+ that involves modulation loop gating rather than channel block as a result of binding in the NT domain.

In the normal brain, cellular types that compose the neurovascular unit, including neurons, astrocytes and endothelial cells express pannexins and connexins, which are protein subunits of two families that form plasma membrane channels. Most available evidence in mammals indicated that endogenously expressed pannexins only form hemichannels, and connexins form both gap junction channels and hemichannels. While gap junction channels connect the cytoplasm of contacting cells and coordinate electrical and metabolic activities, hemichannels communicate intra- and extracellular compartments and serve as diffusional pathways for ions and small molecules. Here, evidence supporting the functional role of hemichannels in the neurovascular unit and white matter under physiological and pathological conditions are reviewed. A sub-threshold acute pathological threatening condition (e.g., stroke and brain infection) leads to glial cell activation, which maintains an active defense and restores the normal function of the neurovascular unit. However, if the stimulus is deleterious, microglia and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins and ATP) that increase the activity of astroglial hemichannels, reducing the astrocyte neuroprotective functions, and further reducing neuronal cell viability. Moreover, ATP is known to contribute to myelin degeneration of axons. Consequently, hemichannels might play a relevant role in the excitotoxic response of oligodendrocytes observed in ischemia and encephalomyelitis. Regulated changes in hemichannel permeability in healthy brain cells can have positive consequences in terms of paracrine/autocrine signaling, whereas persistent changes in cells affected by neurological disorders can be detrimental. Therefore, blocking hemichannels expressed by glial cells and/or neurons of the inflamed central nervous system might prevent neurovascular unit dysfunction and neurodegeneration.

Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.

Mutations in GJB2, which encodes Cx26, are one of the most common causes of inherited deafness in humans. More than 100 mutations have been identified scattered throughout the Cx26 protein, most of which cause non-syndromic sensorineural deafness. In a subset of mutations, deafness is accompanied by hyperkeratotic skin disorders, which are typically severe and sometimes fatal. Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (E1) domains. Here, we examined two such mutations, A40V and G45E, which are positioned near the TM1/E1 boundary and are associated with keratitis ichthyosis deafness (KID) syndrome. Both of these mutants have been reported to form hemichannels that open aberrantly, leading to "leaky" cell membranes. Here, we quantified the Ca(2+) sensitivities and examined the biophysical properties of these mutants at macroscopic and single-channel levels. We find that A40V hemichannels show significantly impaired regulation by extracellular Ca(2+), increasing the likelihood of aberrant hemichannel opening as previously suggested. However, G45E hemichannels show only modest impairment in regulation by Ca(2+) and instead exhibit a substantial increase in permeability to Ca(2+). Using cysteine substitution and examination of accessibility to thiol-modifying reagents, we demonstrate that G45, but not A40, is a pore-lining residue. Both mutants function as cell cell channels. The data suggest that G45E and A40V are hemichannel gain-of-function mutants that produce similar phenotypes, but by different underlying mechanisms. A40V produces leaky hemichannels, whereas G45E provides a route for excessive entry of Ca(2+). These aberrant properties, alone or in combination, can severely compromise cell integrity and lead to increased cell death.

Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of approximately 90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca(2+) concentration ([Ca(2+)](i)). The increase in free [Ca(2+)](i) was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover, inhibition of Cx32 HCs expressed by HeLa cells in low-confluence cultures drastically reduced cell death induced by oxygen-glucose deprivation, which is a more physiological model of ischemia-reperfusion. Thus HC blockade could reduce the increase in free [Ca(2+)](i) and cell death induced by ischemia-like conditions in cells expressing Cx32 HCs.