Myopia Alters the Structural Organization of the Retinal Vasculature, GFAP-Positive Glia, and Ganglion Cell Layer Thickness.

To describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature, and retinal nerve fiber layer thickness, which are critical for inner retinal tissue homeostasis and survival. Astrocyte and capillary distribution, retinal nerve fiber (RNFL), and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01 ± 1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74 ± 0.4D). Untreated marmoset eyes grew normally, their RNFL thickened and their astrocyte numbers were associated with RNFL thickness. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased GFAP-immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels. The myopic changes in retinal astrocytes, vasculature, and retinal nerve fiber layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated.

Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy.

Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.

Human diseases associated with connexin mutations.

Gap junctions and hemichannels comprised of connexins impact many cellular processes. Significant advances in our understanding of the functional role of these channels have been made by the identification of a host of genetic diseases caused by connexin mutations. Prominent features of connexin disorders are the inability of other connexins expressed in the same cell type to compensate for the mutated one, and the ability of connexin mutants to dominantly influence the activity of other wild-type connexins. Functional studies have begun to identify some of the underlying mechanisms whereby connexin channel mutation contributes to the disease state. Detailed mechanistic understanding of these functional differences will help to facilitate new pathophysiology driven therapies for the diverse array of connexin genetic disorders. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome.

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 Ca(2+). This study presents two new possible contributing factors, pH and Zn(2+). Plasma pH levels and micromolar concentrations of Zn(2+) 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 Zn(2+). N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn(2+) 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 Zn(2+). 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.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P(Na)/P(Cl) ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P(Na)/P(GSH) ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Functional effects of Cx50 mutations associated with congenital cataracts.

Mutations in connexin50 (Cx50) cause dominant cataracts in both humans and mice. The exact mechanisms by which mutations cause these variable phenotypes are poorly understood. We have examined the functional properties of gap junctions made by three Cx50 mutations, V44E, D47N, and V79L, expressed in mammalian cell lines. V44E trafficked to the plasma membrane properly and formed gap junctional plaques. However, the mutant did not form functional gap junctions when expressed alone, or with wild-type (WT) Cx46 and Cx50, indicating that V44E is a dominant negative inhibitor of WT connexin function. In contrast, D47N subunits did not localize to junctional plaques or form functional homotypic gap junctions; however, mixed expression of D47N and WT subunits of either Cx50 or Cx46 resulted in functional intercellular channels, with high levels of coupling. Single-channel studies indicated that D47N formed heteromeric channels with WT Cx46 with unique properties. Unlike either V44E or D47N, V79L formed functional homotypic intercellular channels. However, the mutation caused an alteration in voltage gating and a dramatic reduction in the single-channel open probability, resulting in much lower levels of conductance in cells expressing V79L alone, or together with WT connexin subunits. Thus, each mutation produced distinct changes in the properties of junctional coupling. V44E failed to form intercellular channels in any configuration, D47N formed only heteromeric channels with WT connexins, and V79L formed homotypic and heteromeric channels with altered properties. These results suggest that unique interactions between mutant and wild-type lens connexins might underlie the development of various cataract phenotypes in humans.

Sustained Retinal Defocus Increases the Effect of Induced Myopia on the Retinal Astrocyte Template.

The aim of this article is to describe sustained myopic eye growth's effect on astrocyte cellular distribution and its association with inner retinal layer thicknesses. Astrocyte density and distribution, retinal nerve fiber layer (RNFL), ganglion cell layer, and inner plexiform layer (IPL) thicknesses were assessed using immunochemistry and spectral-domain optical coherence tomography on seventeen common marmoset retinas (Callithrix jacchus): six induced with myopia from 2 to 6 months of age (6-month-old myopes), three induced with myopia from 2 to 12 months of age (12-month-old myopes), five age-matched 6-month-old controls, and three age-matched 12-month-old controls. Untreated marmoset eyes grew normally, and both RNFL and IPL thicknesses did not change with age, with astrocyte numbers correlating to RNFL and IPL thicknesses in both control age groups. Myopic marmosets did not follow this trend and, instead, exhibited decreased astrocyte density, increased GFAP+ spatial coverage, and thinner RNFL and IPL, all of which worsened over time. Myopic changes in astrocyte density, GFAP+ spatial coverage and inner retinal layer thicknesses suggest astrocyte template reorganization during myopia development and progression which increased over time. Whether or not these changes are constructive or destructive to the retina still remains to be assessed.

Connexin43 mutation causes heterogeneous gap junction loss and sudden infant death.

BACKGROUND: An estimated 10% to 15% of sudden infant death syndrome (SIDS) cases may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1-encoded gap junction channel protein connexin43 is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases, including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death. METHODS AND RESULTS: GJA1 open reading frame mutational analysis was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole-cell patch-clamp studies were performed to determine the functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white boy and a 3-month-old white girl, respectively. Analysis of the E42K victim's parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in >1000 ethnically matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation, and S272P demonstrated wild-type junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss of function not rescued by wild type. Moreover, the E42K victim's cardiac tissue demonstrated a mosaic immunostaining pattern for connexin43 protein. CONCLUSIONS: This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-connexin43 demonstrated a trafficking-independent reduction in junctional coupling in vitro and a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of connexin43-associated sudden death.

Age exacerbates the effect of myopia on retinal capillaries and string vessels.

The retinal vasculature supplies oxygen and nutrition to the cells and is crucial for an adequate retinal function. In myopia, excessive eye growth is associated with various anatomical changes that can lead to myopia-related complications. However, how myopia-induced ocular growth affects the integrity of the aged retinal microvasculature at the cellular level is not well understood. Here, we studied how aging interacts with myopia-induced alteration of the retinal microvasculature in fourteen marmoset retinas (Callithrix jacchus). String vessel and capillary branchpoint were imaged and quantified in all four capillary plexi of the retinal vasculature. As marmosets with lens-induced myopia aged, they developed increasing numbers of string vessels in all four vascular plexi, with increased vessel branchpoints in the parafoveal and peripapillary retina and decreased vessel branchpoints in the peripheral retina. These myopia-induced changes to the retinal microvasculature suggest an adaptive reorganization of the retinal microvascular cellular structure template with aging and during myopia development and progression.

The N-terminal half of the connexin protein contains the core elements of the pore and voltage gates.

Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.

Gap Junctional Coupling Between Retinal Astrocytes Exacerbates Neuronal Damage in Ischemia-Reperfusion Injury.

Purpose: Retinal astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and unopposed hemichannels. While it is well established that Cx43 is upregulated in retinal injuries, it is unclear whether astrocytic Cx43 plays a role in retinal ganglion cell (RGC) loss associated with injury. Here, we investigated the effect of astrocyte-specific deletion of Cx43 (Cx43KO) and channel inhibitors on RGC loss in retinal ischemia/reperfusion (I/R) injury and assessed changes in expression and GJ channel and hemichannel function that occur in I/R injury. The effect of Cx43 deletion on neural function in the uninjured retina was also assessed. Methods: Cx43 expression, astrocyte density and morphology, and RGC death in wild-type and Cx43KO mice after I/R injury were determined using immunohistochemistry and Western blotting. Visual function was assessed using ERG recordings. GJ coupling and hemichannel activity were evaluated using tracer coupling and uptake studies, respectively. Results: Loss of RGCs in I/R injury was accompanied by an increase of Cx43 expression in astrocytes. Functional studies indicated that I/R injury augmented astrocytic GJ coupling but not Cx43 hemichannel activity. Importantly, deletion of astrocytic Cx43 improved neuronal survival in acute ischemia but did not affect RGC function in the absence of injury. In support, pharmacologic inhibition of GJ coupling provided neuroprotection in I/R injury. Conclusions: The increase in Cx43 expression and GJ coupling during acute I/R injury exacerbates RGC loss. Inhibition of astrocytic Cx43 channels might represent a useful strategy to promote RGC survival in pathologic conditions.

Connexin43 mutations linked to skin disease have augmented hemichannel activity.

Mutations in the gene (GJA1) encoding connexin43 (Cx43) are responsible for several rare genetic disorders, including non-syndromic skin-limited diseases. Here we used two different functional expression systems to characterize three Cx43 mutations linked to palmoplantar keratoderma and congenital alopecia-1, erythrokeratodermia variabilis et progressiva, or inflammatory linear verrucous epidermal nevus. In HeLa cells and Xenopus oocytes, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional gap junction channels with the same efficiency as wild-type Cx43, with normal voltage gating and a unitary conductance of ~110 pS. In HeLa cells, all three mutations also localized to regions of cell-cell contact and displayed a punctate staining pattern. In addition, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D significantly increase membrane current flow through formation of active hemichannels, a novel activity that was not displayed by wild-type Cx43. The increased membrane current was inhibited by either 2 mM calcium, or 5 µM gadolinium, mediated by hemichannels with a unitary conductance of ~250 pS, and was not due to elevated mutant protein expression. The three Cx43 mutations all showed the same gain of function activity, suggesting that augmented hemichannel activity could play a role in skin-limited diseases caused by human Cx43 mutations.

Regulation of connexin hemichannels by monovalent cations.

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.

Optimal lens epithelial cell proliferation is dependent on the connexin isoform providing gap junctional coupling.

PURPOSE: Gap junctions between epithelial cells are essential for normal lens growth. In mice, knockout of Cx50 or targeted replacement of Cx50 with Cx46 (knockin) caused smaller lenses because of decreased epithelial cell proliferation. However, it remains unclear whether Cx50 functionally contributes to lens epithelial coupling during maximal proliferation on postnatal day 2 (P2) and P3. To determine which connexins functionally contribute to epithelial cell coupling and proliferation, junctional coupling from epithelial cells of wild-type and knockin mice was examined. METHODS: Epithelial cells were isolated from wild-type or knockin mice at different developmental ages. Junctional currents were measured by dual whole cell voltage clamp. Cell proliferation was assayed by BrdU incorporation. Connexins were immunolocalized using specific antibodies. RESULTS: Junctional currents between lens epithelial cells exhibited a developmentally regulated sensitivity to quinine, a drug that blocks Cx50 gap junctions, but not Cx43 or Cx46. Single-channel currents had a unitary conductance of 210 pS, typical of Cx50. Immunocytochemical staining showed Cx43 and Cx50 were abundantly expressed in wild-type cells, and Cx46 replaced Cx50 in knockin cells. A correlation between functional activity of Cx50 and maximal proliferation was also found. In epithelial cells from P3 wild-type mice, there was a high density of BrdU-labeled nuclei in both the central epithelium and the equatorial epithelium, and 60% or more of total coupling was provided by Cx50. In older cells, proliferation was greatly reduced, and the contribution of Cx50 to total coupling was progressively reduced (45% or less on P12; 25% or less on P28). Coupling between epithelial cells of Cx46 knockin mice was similar in magnitude to that of wild-type mice but had pharmacologic and biophysical characteristics of Cx46. This functional replacement of Cx50 with Cx46 was correlated with 71% and 13% reductions in BrdU-labeled cells in the P3 central epithelium and equatorial epithelium, respectively. CONCLUSIONS: These results reconcile previous genetic studies showing that Cx50 influences epithelial cell proliferation, with numerous studies suggesting that Cx43 was the principal epithelial cell connexin. They further show that the contribution of Cx50 is highest during peak postnatal proliferation but progressively declines with age thereafter.

Functional characterization of a naturally occurring Cx50 truncation.

PURPOSE: Lens connexins undergo proteolytic cleavage of their C termini during fiber maturation. Although the functional significance of this is unknown, cleavage has been correlated with changes in channel-gating properties. This study evaluates the functional consequences of this endogenous truncation by characterizing the properties of a C-terminal truncated Cx50 protein. METHODS: Murine and human Cx50 were truncated at amino acids 290 and 294, respectively, before expression in paired Xenopus oocytes or mammalian cells. Protein expression was evaluated by immunocytochemistry. Dual whole-cell voltage clamp techniques were used to analyze macroscopic and single-channel conductance, voltage-gating properties, and kinetics; pH gating sensitivity was measured by superfusion with 100% CO2-saturated media. RESULTS: Cx50tr290 channels exhibited an 86% to 89% reduction in mean macroscopic conductance compared with full-length Cx50. Heterotypic channels formed functional gap junctions, displayed an intermediate level of coupling, and exhibited unaltered voltage-gating properties. C-terminal truncation did not alter single-channel gating characteristics or unitary conductance. Interestingly, truncated and full-length Cx50 channel conductances were reversibly blocked by cytoplasmic acidification. CONCLUSIONS: C-terminal truncation of Cx50 did not inhibit the formation of homotypic or heterotypic channels. However, a significant decrease in conductance was observed for truncated channels, a phenomenon independent of alterations in voltage-gating sensitivity, kinetics, or chemical gating. These results provide a plausible explanation for the 50% decrease in junctional coupling observed during lens fiber maturation.

New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43.

Aquaporin-4 (AQP4), the main water channel in the brain, is expressed in the perivascular membranes of mouse, rat, and human astrocytes. In a previous study, we used small interfering RNA (siRNA) to specifically knock down AQP4 in rat astrocyte primary cultures and found that together with reduced osmotic permeability, AQP4 knockdown (KD) led to altered cell morphology. However, a recent report on primary cultured astrocytes from AQP4 null mice (KO) showed no morphological differences compared with wild types. In this study, we compared the effect of AQP4 KD in mouse, rat, and human astrocyte primary cultures and found that AQP4 KD in human astrocytes resulted in a morphological phenotype similar to that found in rat. In contrast, AQP4 KD in mouse astrocytes caused only very mild morphological changes. The actin cytoskeleton of untreated astrocytes exhibited strong species-specific differences, with F-actin being organized in cortical bands in mouse and in stress fibers in rat and human astrocytes. Surprisingly, as a consequence of AQP4 KD, F-actin cytoskeleton was depolymerized in rat and human whereas it was completely rearranged in mouse astrocytes. Although AQP4 KD induced alterations of the cell cytoskeleton, we found that the expression of dystrophin (DP71), beta-dystroglycan, and alpha-syntrophin was not altered. AQP4 KD in cultured mouse astrocytes produced strong down-regulation of connexin43 (Cx43) with a concomitant reduction in cell coupling while no major alterations in Cx43 expression were found in rat and human cells. Taken together, these results demonstrate that with regard to these properties, human astrocytes in culture are more similar to rat than to mouse astrocytes. Moreover, even though AQP4 KD in mouse astrocytes did not result in a dramatic morphological phenotype, it induced a remarkable rearrangement of F-actin, not related to disruption of the dystrophin complex, indicating a primary role of this water channel in the cytoskeleton changes observed. Finally, the strong down-regulation of Cx43 and cell coupling in AQP4 KD mouse astrocytes indicate that a functional relationship likely exists between water channels and gap junctions in brain astrocytes.

Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels.

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 Ca(2+) 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.

Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens.

PURPOSE: Many proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements. METHODS: Human lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)-electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells. RESULTS: Cleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels. CONCLUSIONS: Accumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.

Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine.

Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 µM), using an extracellular divalent cation, zinc (Zn(++)), as a nonspecific positive control for comparison (IC50∼3 µM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

Prospects for rational development of pharmacological gap junction channel blockers.

Connexin-null mice and human genetic gap junction diseases illustrate the important roles that gap junction channels play under normal conditions, and the neuro- and cardioprotective effects of gap junction blocking agents demonstrate that closure of these channels may be beneficial in certain pathological situations. This overview summarizes studies in which gap junction modifying reagents have been characterized, highlighting examples of agents for which selectivity for gap junction subtypes has been demonstrated. In addition, strategies for targeting connexin domains through peptide inhibitors are outlined, which may ultimately provide agents that are not only connexin-selective in their actions, but also affect only a subset of a gap junction channel's gating responses.