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(The FASEB Journal. 2006;20:2329-2338.)
© 2006 FASEB

Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels

Daniel González, Juan M. Gómez-Hernández and Luis C. Barrio1

Unit of Experimental Neurology Research Department, "Ramón y Cajal" Hospital, Madrid, Spain

1Correspondence: Unidad de Neurología Experimental, Departamento de Investigación, Hospital "Ramón y Cajal," Carretera de Colmenar km 9, 28034-Madrid, Spain. E-mail: luis.c.barrio{at}hrc.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations of connexin-26 (Cx26) cause nonsyndromic hearing loss and other syndromes affecting ectoderm-derived tissues. While the exact mechanisms underlying these diseases remain elusive, Cx’s are generally considered to mediate cell-to-cell communication by forming gap junction channels. We show here that unlike rat Cx26, human and sheep Cx26 form voltage-gated hemichannels when expressed in oocytes and Neuro2A cells. A single evolutionary amino acidic change at position 159 of the rodent protein, the replacement of aspartic acid with asparagine in the human and sheep proteins, accounts for this species specificity. At the resting potential and in normal millimolar extracellular calcium, open human Cx26 hemichannels can be detected both electrophysiologically and by dye uptake, although they did not affect cell viability. These hemichannels opened at ~ –50 mV and their activation increased by depolarization until they inactivate at positive membrane potentials. Single-channel analysis revealed that activation and inactivation involved two distinct voltage gating mechanisms and that the fully open hemichannel displays a conductance twice that of the intercellular channel. The existence of a hemichannel that opens under physiological control of the membrane potential may have important implications for the normal and pathological activity of Cx26 in humans, particularly with respect to hearing and the epidermis.—González, D., Gómez-Hernández, J. M., Barrio, L. C. Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels.


Key Words: GJB2 • hearing • skin • deafness • genodermatosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CONNEXINS (CX) belong to a multigene family that typically forms intercellular channels. In humans there are at least 20 members, and most Cx orthologues have been identified in other vertebrate species (1) . The assembly of connexins into gap junction channels occurs in two main stages (2) . Connexins first oligomerize into hexameric hemichannels prior to reaching the cell surface, then two hemichannels from neighboring cells dock to generate a complete intercellular channel. However, undocked hemichannels at the cell surface can also open on membrane depolarization, as initially described for Cx46 (3) . Indeed, there is now considerable evidence that the hemichannels of several different connexin proteins are capable of being electrically and chemically activated even in native cells (4) . Open hemichannels can produce a rapid flow of ions and ATP, NAD+, glutamate, and prostaglandins across cell membranes (5 6 7 8 9) . Hence, they have been implicated in an increasing number of physiological and pathological processes (10 , 11) . However, not all Cx isoforms may form hemichannels that open, and this property may not even be conserved between orthologous Cx’s, e.g., the neuronal Cx36 (12 , 13) . While open human Cx26 hemichannels have been observed in transfected HeLa cells (14) and Xenopus oocytes (15 , 16) , there is little information regarding the properties of rat or mouse Cx26 hemichannels. It is known that Cx26 hemichannels in the horizontal cells of the teleost and turtle retina are involved in an electrical feedback mechanism that regulates glutamate release from photoreceptors (17 18 19) . Furthermore, mammalian Cx26 actively participates in the physiology and pathology of a variety of organs including liver, kidney, intestine, lung, spleen, testes, brain (20) , mammary gland (21) , cochlea (22 , 23) , and skin (24 25 26) . Thus, it is important to consider the influence that Cx26 hemichannels might have on these tissues. In humans, the importance of Cx26 in the inner ear and epidermis is underlined by the fact that mutations in this gene are the most common cause of hearing loss of genetic origin and that specific dominant Cx26 mutations also result in epidermal disorders (27; http://www.crg.es/deafness). Here we show that while human and sheep Cx26 hemichannels are subject to voltage gating, those expressed by the rat are not. We demonstrate that a single amino acid substitution in the rat sequence is responsible for this species-specific loss of gating. Significantly, the electrophysiological characterization of human Cx26 hemichannels revealed that the activation and the distinct conductance states of these hemichannels were strictly regulated by the degree of membrane depolarization through two distinct mechanisms of voltage gating. The existence of functional human Cx26 hemichannels under physiological control of the membrane potential provides a rational basis to elucidate the function of Cx26 in humans and the hemichannel involvement in the pathogenesis of deafness and skin disorders caused by mutations within the Cx26 gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular cloning
The coding region of the human (h) and sheep (s) Cx26 was amplified from genomic DNA with primers corresponding to the amino- and carboxyl-terminal sequences, and polymerase chain reaction (PCR) products were subcloned into the pBSXG transcription vector (28) . The hCx26 and sCx26 cDNA sequences were identical to those available in GenBank (NM_M86849 and NM_U17592). The rat (r) Cx26 cDNA is inserted into the pSp64T plasmid (29) . To express rCx26 and hCx26 in mammalian cells, the cDNAs were subcloned into the bicistronic pIRES2-enhanced GFP expression vector (Clontech, Palo Alto, CA, USA). The N159D substitution in rCx26 was created by PCR mutagenesis and confirmed by DNA sequencing. The primers used for mutagenesis were sense 5'-CAGCGACTAGTGAAGTG TAAC-3' and antisense 5'-CATGAAGAAGCCATCGTACAT-3'.

Expression systems
The preparation of Xenopus oocytes and RNA microinjections was performed as described previously (29) . Oocytes were injected with an antisense oligonucleotide (15 ng/oocyte) that blocks endogenous Cx38 expression (29) , either alone (controls) or with Cx26 mRNAs (0.5–1.0 µg/µl, 50 nl/oocyte). To allow intercellular channels to form, pairs of oocytes were brought into contact after removing the vitelline membrane. The murine neuroblastoma 2A cell line [Neuro2A; American Type Culture Collection (ATCC), CCL131] was cultured in 35 mm dishes at 37°C in a moist atmosphere containing 5% CO2 and in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FCS, 4.5 g/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown to 60–80% confluence, then lipofectamine transfected with the pIRES-enhanced GFP vectors, with or without the hCx26 and rCx26 inserts. The day before recording, the cells were plated in quadruplicate at low density (5x104 cells per 35 mm dish) to impair gap junction formation.

Electrophysiology
The transmembrane currents (Im) of single oocytes were recorded by the conventional two-electrode voltage clamp method (28) . Junctional currents (Ij) between oocyte pairs were measured using the dual voltage-clamp technique (29) . Single-channel recording of hemichannels in oocytes and Neuro2A cells was performed in the cell-attached configuration using standard patch-clamp techniques (28) . Pipettes for oocyte patches were filled with external ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2 and 5 HEPES, pH 7.5) or standard internal solution (SIS; in mM: 100 KCl, 10 HEPES, 10 EGTA, pH 7.2). The bath solution used was SIS since under these conditions the resting membrane potential was near zero, as confirmed by the similarity of the unitary conductance when the cell-attached patch was excised at the end of recordings. Enhanced GFP (EGFP) was visualized in transfected Neuro2A cells by fluorescence microscopy and these cells were targeted for unitary recordings. The recording chamber was superfused with modified Krebs-Ringer solution containing (in mM) 140 NaCl, 4 KCl, 2 CsCl, 2 CaCl2, 1 MgCl2, 1 BaCl2, 2 pyruvate, 5 Glc, 5 HEPES, pH 7.4, and 310 mOsm; patch pipettes were filled with the same external solution. Macroscopic and unitary currents were filtered at 200 Hz and 1 kHz, and sampled at 1 kHz and 4 kHz, respectively. For data acquisition and analysis we used a Digidata interface and pClamp (Axon Instruments, Inverurie, Scotland), as well as Sigmaplot software (Jandel Scientific, Corte Madera, CA, USA). The Student’s t test was used for statistical analysis.

Fluorescent imaging
Fluorescence-activated cell scanning (FACS) analysis was performed with the FACSCalibur system and CELLQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). Neuro2A cells were harvested from one 35 mm dish with the culture medium, collecting all live and dead cells. Data acquisition was terminated when 20,000 events were counted and debris was clearly discriminated from cells on the basis of size and complexity. EGFP-positive and negative cells were efficiently selected by 488 nm line excitation at 530/30 nm emission and the statistics were calculated. Propidium iodide dissolved in DMEM (PI; 100 µM) was added to the cells for 1 min and 1 h prior to FACS analysis. For each sample, 10,000 EGFP-positive cells were analyzed and the subpopulation of red PI-positive fluorescent cells was detected at 635 nm line excitation at 585/42 nm. For dye uptake experiments in oocytes, 1 mM PI dissolved in ND96 was added for 30 min. After washout, confocal images of oocytes (x10 objective; MRC-1024, Bio-Rad, Hercules, CA, USA) and of the transfected Neuro2A cells (x100 oil) were collected at emission wavelengths of 598/40 nm using 658 nm line excitation.

Immunohistochemistry
Neuro2A cells were fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 30 min and permeabilized with ethanol:acetic acid (95% v/v) for 10 min at –20°C. Cells were preincubated in a blocking solution (10% goat serum in PBS) and incubated with primary antibodies against Cx26 (Cat. 71–0500, Zymed Laboratories, San Francisco, CA, USA; 1:500) at 4°C overnight. After washing, the cells were incubated in CyTM5-conjugated goat anti-rabbit IgG antibody (Ab) (Cat. 81–6116, Zymed; 1:100). Confocal z-serial 1 µm images were collected at 680/32 nm emission using the 647 nm line excitation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human and sheep but not rat Cx26 form open hemichannels
When cRNA’s encoding for Cx26 proteins were expressed in oocytes, those injected with human but not those with the rat Cx26 developed a progressive depolarization of the resting potential paralleled by an increase in membrane conductance in normal ND96 solution (1.8 mM Ca2+ and 1 mM Mg2+; Fig. 1 A, B). Upon switching to the voltage-clamp mode, large activated inward currents were detected only in hCx26 injected oocytes (Fig. 1C ). These currents declined slowly at a holding potential of –80 mV until they fully deactivated. Subsequent application of repetitive depo-larizing pulses to +40 and +80 mV induced slowly activating outward currents that became inward and in turn deactivated slowly during the repolarization phase of the pulses at –80 mV (Fig. 1E ). Two forms of blocking connexin hemichannels/channels—acidification of the bath solution with 100% C02 (pH 6.0) and exposure to 100 µM carbenoxolone—suppressed the activation of these currents. Furthermore, oocytes expressing hCx26 but not rCx26 were also permeable to propidium iodide (PI; Fig. 1F ); the uptake of PI was also blocked by carbenoxolone, consistent with its entry through the hemichannels. Taken together, these data indicate that hCx26 but not rCx26 is able to form voltage-gated hemichannels under normal conditions (i.e., at a resting membrane potential and normal extracellular divalent cation concentrations). In contrast, rCx26 hemichannels remained closed until oocytes were brought into contact and gap junction channels acquired the open conformation (Fig. 1D ).


Figure 1
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  		Figure 1. The human but not the rat Cx26 forms voltage-gated hemichannels. Xenopus oocytes were injected with equal amounts of hCx26 or rCx26 mRNA, along with antisense mRNA to block endogenous Cx38, and they were then incubated in ND96 solution (1.8 mM Ca2+ and 1 mM Mg2+). Control oocytes were injected with Cx38 antisense mRNA alone. Only hCx26 oocytes showed a progressive depolarization of the resting potential (A), a reduction of the input membrane resistance (B), and exhibited large transmembrane currents (C). h.a.i., hours after injection. D) In the same experiments, hCx26 and rCx26 oocyte pairs but not control oocytes developed high levels of electrical coupling. Each point in panels A–D is the mean value ±SE of 15–20 measurements from 3 experiments. E) The application of voltage pulses in hCx26 oocytes (of 10 s and 0.03 Hz) from holding at –80 mV to +40 mV (upper) or +80 mV (bottom) induced activating slowly outward currents that, upon returning to –80 mV, became inward and slowly declined until they fully closed. The currents attributed to the activation of hemichannels were completely blocked by CO2 acidification (upper, 1–2) and carbenoxolone (bottom, 1–2). F) After 30 min in the presence of 1 mM propidium iodide, hCx26 oocytes took up the dye (as seen in confocal images), unlike rCx26 oocytes or hCx26 oocytes treated with carbenoxolone. Scale bars, 200 µm.

The difference between human and rat Cx26 in terms of functional hemichannel formation was further confirmed in transiently transfected Neuro2A cells. More than 65% of transfected rCx26- and hCx26-tranfected cells were immunostained with anti-Cx26 antibodies (Fig. 2 A). In both cases, z-serial confocal images revealed a similar pattern of fine punctuate labeling in the plasma membrane, which probably corresponds to hemichannels that have been delivered to the cellular surface (30) . Plaques could also be observed between apposed cells, probably representing gap junction complexes. Upon FACS analysis, no difference in the percentage of cell death was observed in Neuro2A cells transfected with the pIRES-enhanced GFP vector alone or that containing the rCx26 and hCx26 inserts (Fig. 2B, C ). However, the percentage of hCx26 transfected cells that took up PI after 60 min in normal medium (1.8 mM Ca2+ and 0.8 mM Mg2+) was 7-fold greater than in controls (5.0±2.8 to 35.7±10.9%; P<0.05), and this increase was sensitive to 100 µM carbenoxolone (Fig. 2C ). In contrast, PI uptake was no greater in rCx26 cells than in controls. In confocal images of hCx26 cells, PI was uniformly distributed in the cytoplasm and nucleus as opposed to the characteristic DNA staining of apoptotic or necrotic cells with membrane damage (Fig. 2D ). Furthermore, voltage-gated hemichannels were found in >55% of recordings from hCx26-transfected cells expressing EGFP (n=110) but not in cells expressing rCx26 (n=52) or control (n=36) cells. As in oocytes, open hCx26 hemichannels were detected in Neuro2A cells under resting conditions without causing adverse effects on cell viability.


Figure 2
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  		Figure 2. Open human Cx26 hemichannels were detected under resting conditions and they did not affect cell viability. Neuro2A cell cultures were transfected with the p-IRES-enhanced GFP vector alone (enhanced GFP) or that containing the hCx26 and rCx26 insert. A) Immunostaining of hCx26 and rCx26 cultures 24 h post-transfection. Confocal z-serial images (1, 2) from cell pairs (a, b) showing fine punctate staining at the cell surface (1, arrowheads), plaques at the cell-cell contact areas (2, arrows), and, less frequently, pronounced cytoplasmic labeling (2, asterisk). B) FACS analysis using EGFP as a reporter of expression. No differences were observed in the percentage of green fluorescent cells in each culture and the mean intensity per cell (insert; in arbitrary units, AU) 24–96 h post-transfection. C) Percentage of red and green fluorescent cells after labeling with 100 µM propidium iodide in normal medium (1.8 mM Ca2+ and 0.8 mM Mg2+) for 1 min, indicative of cell death, or when incubated for 1 h in the dye uptake assay (48 h post-transfection). Little cell death was observed in the three cultures analyzed. After extended exposure to PI, the percentage of red cells only increased in hCx26-transfected cells (asterisk; t test P<0.05), and this effect was blocked by carbenoxonole. The data in panels B and C represent the mean values ±SE from 4 experiments. D) Confocal analysis of the uptake experiments in panel C. The images show 1) all cells present in the culture under bright-field phase contrast; 2) those cells positively transfected with rCx26 or hCx26 insert are stained green, and 3) cells loaded with PI are red. HCx26 but not rCx26 expressing cells took up PI, which was distributed uniformly in the cytoplasm and nucleus. A, D) Scale bars, 10 µm.

Voltage dependency of mammalian Cx26 hemichannels: molecular determinants
The activity of hCx26 hemichannels is strictly controlled by the membrane potential (Fig. 3 , Fig. 4 , Fig. 5 ). Macroscopically, the activation of hemichannels in oocytes was detected with 10 s –60/–40 mV pulses applied from a holding potential of –80 mV, and it increased progressively in accordance with the degree of membrane potential depolarization (Fig. 3A, B ). However, at higher positive potentials the currents decreased (>+40mV), indicating that hCx26 hemichannels were inactivated. Currents flowed inwardly at negative potentials, then outwardly, crossing the reversal potential near zero (~–5mV). The currents displayed a slow time course during activation and deactivation, on the scale of seconds, whereas inactivation was more rapid (see supplemental Fig. 7, Table 1). Currents with such characteristics were not elicited from rCx26 oocytes in normal external solution (Fig. 3A ) or in low Ca2+ conditions (0.5 mM; data not shown). However, currents that could be attributed to the opening of hemichannels on depolarization were also registered in oocytes injected with sheep Cx26 (sCx26). The opening of sCx26 hemichannels was detected at more positive potentials (~–20mV) and the currents increased monotonically with the membrane depolarization across the entire voltage range explored (Fig. 3A, B ).


Figure 3
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  		Figure 3. Voltage dependency of human, sheep, and rat Cx26 hemichannels. A) Sample records of transmembrane currents (Im) evoked in ND96 solution (1.8 mM Ca2+ and 1 mM Mg2+) by the application of voltage pulses (Vm) from a holding of –80 mV to +80 mV, in increments of 20 mV and of 10 s duration. Upon depolarization, slowly activating currents were detected in hCx26 and sCx26, but not in rCx26 oocytes. Introduction of the Asn159Asp mutation into the rat sequence (rCx26-N159D) rescued hemichannel function. B) Ghj/V relationships. Hemichannel conductance (Ghj) measured at the end of the 10 s pulses was calculated for the reversal potential of –5 mV, then normalized relative to its maximal value in each oocyte. Activation of hCx26 and rCx26-N159D mutant hemichannels was detected at ~ –50 mV and as the degree of depolarization progressed, the conductance first increased before decreasing at higher positive potentials (see arrows in panel A; see also Figs. 7 and 9 in supplemental material). The opening of sCx26 hemichannels shifts toward more positive potentials and the conductance increases monotonically upon depolarization across the whole voltage range were explored. The solid line is the Boltzmann fit determined separately for activation and inactivation phases. Parameters of fitting are listed in supplemental Table 1. Each point represents the mean value ± SE (n=10 from 3 different experiments).


Figure 4
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  		Figure 4. Voltage dependency of single human Cx26 hemichannels. Example of a cell-attached patch recording in transfected Neuro2A cells with 2 mM Ca2+ and 1 mM Mg2+ outside. A) Slow depolarizing voltage ramps (1 min duration and with 3 min in between) from –80 to +80 mV typically induced the opening of single hemichannels in the patch to {gamma}1=432 pS and {gamma}2=877 pS at moderate depolarizations. The hemichannels then stayed open most of the time until they closed at high positive potentials. Unitary currents flow inwardly at negative potentials, then become outward by crossing the reversal potential of ~ –7 mV. Currents were corrected for leakage by subtracting the residual currents linearly extrapolated from the full closing transitions. B) Po/V relationship. The open probability (Po) was calculated from ensemble averaging of currents for 12 sequential ramps normalized relative to the conductance value of the two hemichannels simultaneously opened. The Po was maximal at ~ +40 mV and it decreased asymmetrically toward more negative and positive potentials (cf., Fig. 3B ). The solid line is the Boltzmann fit determined separately for activation and inactivation phases. Fitting parameters are listed in supplemental Table 1.


Figure 5
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  		Figure 5. Activation and inactivation of hCx26 hemichannels involve two distinct forms of voltage gating. Example of a cell-attached patch recorded from an hCx26 oocyte with 1.8 mM Ca2+ and 1 mM Mg2+ in the pipette. A) Unitary responses to voltage steps of 40 s applied from a holding potential of –80 mV to +40, +60, and +80 mV and the corresponding all-points histograms. B) Typically, at +40 mV a single hemichannel first opened several seconds after the pulse onset (arrowhead), and this normally was preceded by a flickering activity (asterisk). Subsequently, the hemichannel spent most of the time in the main open state (o) of {gamma}o = 316.71 ± 12.15 pS (Po=0.58). Transition to the closed state (c) or to a residual open state (r) of {gamma}r = 34.64 ± 6.05 pS was infrequent (Pc=0.19 and Pr=0.07). Note that in the patch a second hemichannel opened at the end of the 40 s pulse (O2). With increasing voltages, the time spent in the fully open state decreased (Po=0.20 at+60 and Po=0.06 at+80) whereas in the residual open state, the time increased (Pr=0.40 at +60 and Pr=0.81 at +80).

Because of the close identity between rat, sheep, and human Cx26 sequences (90.7%), it seemed likely that the divergence of only a few amino acids might be responsible for the loss of voltage gating in rat hemichannels (see amino acid alignment in the supplemental Fig. 8). Mutational analysis revealed that replacing the single polar uncharged Asn at position 159 of rCx26 with the negatively charged Asp present in hCx26 and sCx26 was sufficient to recover the gating function (Fig. 3A, B ). The rCx26-N159D mutant formed hemichannels with voltage gating properties that closely resembled those of hCx26. Moreover, rCx26-N159D hemichannels were permeable to PI, showed inactivation at high positive potentials, and the unitary properties were similar to those described below for hCx26 hemichannels (see supplemental Fig. 9). Indeed, the mutant rCx26-N159D retained the capacity to form channels and did not modify voltage dependence properties of junctional conductance (see supplemental Fig. 10, Table 2).

Unitary recordings of hCx26 hemichannels revealed two distinct forms of voltage gating depending on the polarity of the membrane potential (Fig. 4 , Fig. 5 ). In cell-attached patches of hCx26-tranfected cells with a normal extracellular divalent cation concentration (2 mM Ca2+ and 1 mM Mg2+), application of slow depolarizing voltage ramps (1 min) from a holding potential of –80 mV to + 80 mV typically induced the opening of single hemichannels at negative potentials (~ –50 mV). As depolarization progressed, these channels remained stable in a high conductance open state until polarization reached high positive potentials, at which point the single hemichannels closed (Fig. 4A ). The open probability (Po), calculated as the ensemble averaging of unitary currents along the ± 80 mV voltage ramp, correlated well with the bell-shaped curve of macroscopic conductance. The maximum Po was centered at +40 mV with asymmetric falls toward more negative and positive voltages (Fig. 4B ). Cell-attached patch recordings under steady-state conditions were performed on hCx26 oocytes (Fig. 5A ). In accordance with the slow activating time course of macroscopic currents, the initial transition from the closed state to the main open state upon applying voltage steps from –80 to +40 mV was normally delayed with respect to the onset of the voltage pulse, in the time scale of seconds. Furthermore, this transition was generally preceded by a characteristic flickering activity. Once the hemichannel had been activated, it remained open for most of the remaining 40 s pulse (Po=0.58), in accordance with the generation of the peak current around +40 mV at both the macroscopic and unitary levels (see Fig. 3B , Fig. 4A ). Moreover, the conductance at the main open state was 316.71 ± 12.15 pS ({gamma}o), about twice the conductance of the intercellular channel (31) , suggesting that it corresponds to the fully open hemichannel. At +40 mV, the transitions from the fully open state to the closed state (c) or to other conductance sublevels, such as a residual open state (r) of {gamma}r= 34.64 ± 6.05 pS, rarely occurred (Pc=0.19 and Pr=0.07, respectively). As the voltage increased to +60 and +80 mV, the latency and flickering activity preceding the first opening was reduced and the time spent in the fully open state decreased from a Po = 0.58 at +40 mV to 0.20 and 0.06, respectively. Furthermore, the time in the residual open state augmented from Pr = 0.07 at +40 mV to 0.40 and 0.81, whereas time spent in the closed state did not vary. Thus, while the dominant gating at negative potentials involved a transition between the closed and the fully open high conductance state, the mechanism responsible for inactivation at positive voltages caused incomplete closure of fully open hemichannels to a residual open state of ~9-fold smaller conductance. Transitions between these three principal intervening states occurred rapidly (<1–2 ms) or less frequently, resulting in an elongation of the flickering mode. Unitary I/V relationships measure at symmetrical KCl concentrations showed that the current flowing through the fully open hCx26 hemichannel was slightly inward rectifying, adopting a linear behavior of ~450 pS at negative potentials and a sublinear behavior of ~320 pS at positive voltages (Fig. 6 ).


Figure 6
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  		Figure 6. Unitary I/V relationship of hCx26 hemichannels. Oocyte patch recording was performed in the cell-attached configuration with symmetrical KCl inside/outside concentration. A single hemichannel was first activated at +40 mV for 10 s, then 10 sequential 100 ms voltage ramps over ±80 mV were applied. Current flowing throughout the fully open hemichannel rectified slightly, varying the conductance from ~450 pS at negative potentials (continuous line) to ~320 pS at positive potentials (dotted line).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrate that human and sheep Cx26, but not the rat orthologue, are able to form open, voltage-activated hemichannels. We also show that rat hemichannels became voltage-gated when the Asn at position 159 of the rat sequence was replaced by the Asp found in the human and sheep. Residue N159/D159 lies in the second extracellular loop, near the boundary with the third transmembrane helix, where the molecular components of the so-called "gating loop" have been provisionally assigned (5) . This gating "loop" is responsible for the transitions from the closed to the fully open state on moderate depolarization of the membrane potential. We also identified a distinct gating mechanism that is involved in the process of hemichannel inactivation, since it causes incomplete closure from the fully open state to a residual open state at high positive potentials. Indeed, a similar two-gate model at opposite polarities has been described in the rat Cx46 and the mouse Cx30 and Cx50 hemichannels (5 , 32 , 33) . In human Cx32 hemichannels, these gates act at the same voltage polarity (28) . The gating responsible for the inactivation of undocked hCx26 hemichannels may correspond to that of gap junction channels regulated by transjunctional voltage since both operate at a positive internal polarity between the equivalent intervening states (34 , 35) .

During their evolution, the primary sequences of human, sheep, and rat Cx26 orthologues have not received insertions or suffered deletions, and only 19 residues of the consensus 226 a.a. have changed between these three mammalian species (see supplemental Fig. 8). However, the degree of identity between human and sheep Cx26 that are able to make functional hemichannels is somewhat higher than the identity to the rat Cx26. Nevertheless, there are only 10 residues in the human and sheep sequences that diverge from that of the rat, most of which are conservative substitutions. However, there are two clear exceptions: the Asp159 to Asn and the Ala197 to Ser substitutions. By mutating these two residues in the rat, we found that only the N159D substitution induced the formation of voltage-activated open hemichannels. Since the N159D mutation introduces a negatively charged residue in exchange for a polar uncharged residue, the Asp159 would be an integral part of the voltage sensor. The neutralization of Asp159 in human Cx26 (D159N), the inverse mutation of N159D in the rat, did not eliminate the voltage activation of hemichannels, whereas reverting the polarity of the charge (D159R) suppressed the gating of the hemichannel without affecting its formation or the voltage regulation of the intercellular channels (data not shown). Therefore, the rescue of the gating mechanism in the rat by the N159D substitution might be due to a structural change that results from the interaction of Asp with other adjacent residues or with those located in neighboring domains. In this context, the H161N mutation in mouse Cx50, located within the third transmembrane domain, also eliminates the activity of the hemichannel without affecting channel formation or its transjunctional voltage dependence (36) .

Although the presence of hemichannels at the cell surface constitutes a normal phase in the life cycle of gap junction proteins, prolonged depolarization of membrane potential at high positive potentials and a reduction in the external calcium concentration are necessary to open most functional hemichannels (4) . However, the human Cx26 hemichannels belong to that restricted subset of hemichannels that are capable of opening at the resting membrane potential and with millimolar extracellular calcium. Moreover, our electrophysiological analysis revealed that the activation of hCx26 hemichannels is strictly regulated by the membrane potential. Hence, voltage dependency within a physiological range would appear to be the most likely mechanism to control hCx26 hemichannel activity under normal calcium and pH conditions (15) . In addition, the voltage-dependent inactivation of hCx26 hemichannels may be capable of preventing the full activation of hemichannels upon prolonged depolarization of the membrane potential at high positive potentials, maintaining them in a residual open state with ~9-fold smaller conductance. Given the probabilistic nature of voltage-dependent opening of hCx26 hemichannels, the expression of these hemichannels might be expected to be deleterious, as reported in oocytes and transfected cells that undergo lysis and cell death (15 , 16 , 37) . However, hCx26 hemichannels are remarkably well tolerated without affecting cell viability possibly due to a lower abundance, as in this study and other previous reports (e.g., refs. 14 , 38 ).

The permeabilization characteristics of hCx26 hemichannels are those expected of gap junction channels. The conductance value of fully open hemichannel and of the residual open state is about twice that of the complete channels (31) . The current flowing through the hemichannels at macroscopic and unitary levels reverses at a membrane potential near zero (~ –5 mV). Furthermore, the reversal potential does not change when measured with external NaCl or KCl, indicating that it is nonselective for monovalent cations. Under symmetrical KCl concentrations, the unitary inward current flow at negative potentials is somewhat greater (1.4-fold) than that in the outward direction for positive potentials. This difference probably reflects the slight preference for cations described for rat Cx26 channels (39) . Like the intercellular channel (38) , hCx26 hemichannels are permeable to cationic tracers such as propidium iodide (MW 668, +2 charges; in this study) and the anionic dye Lucifer yellow (MW 443, –2 charges), and it can release ATP into the extracellular medium (14) .

The capacity of human Cx26 to form voltage-gated hemichannels that could be activated upon moderate depolarization of membrane potentials at normal extracellular [Ca2+] may be relevant to the physiology and pathology of several tissues. Cx26 is important in auditory function, and in the rat/mouse and human cochlea it is highly expressed in two independent networks of coupled cells: the epithelial cell system and the connective cell system (22 , 40 , 41) . In this structure, Cx26 is thought to play a critical role in the rapid removal of K+ away from the base of the hair cells and in recycling K+ back to the endolymph (42 , 43) . The extracellular accumulation of K+ ions at the basolateral surface of hair cells is highly deleterious and, accordingly, the loss of Cx26 gap junctions from the inner ear epithelial network in the Cx26Otog-Cre mice causes deafness and the death of supporting and hair cells at the onset of hearing activity (23) . A similar pathogenic mechanism may be involved in the recessive deafness-linked Cx26 mutations tested so far in which cell-to-cell communication is disrupted (e.g., ref. 44 ). Furthermore, in another group of pathological mutations that are able to form functional channels, the metabolic coupling mediated by Ins(1,4,5)P3 was specifically disrupted without affecting ionic permeability (45 , 46) . In this context, it seems plausible that the opening of human Cx26 hemichannels in response to high K+-induced depolarization could contribute to spatial K+ buffering. These hemichannels may mediate ion flow across the plasma membrane, particularly at the points of the recycling pathway where intracellular transfer through gap junctions is disrupted (e.g., between the hair cells and the supporting cells). Because active hCx26 hemichannels enable ATP to be released into the extracellular medium (14) , they will also participate in purinergic intercellular signaling in the cochlea. Thus, the ATP released via hCx26 hemichannels can mediate the recently proposed negative feedback between supporting cells and outer hair cells to control hearing sensitivity, with a protective role at high intensities (47) . Alternatively, ATP may act in the neighboring supporting cells to spread the propagation of intercellular Ca2+ waves that seem to be required to maintain the ionic homeostasis of the cochlear fluids (48) .

Connexin26 is also thought to play a critical role in controlling keratinocyte growth and differentiation and in maintaining the homeostasis of the epidermis. Indeed, certain deafness-linked Cx26 mutations are accompanied by skin disorders (49) . In the epidermis, the species specificity of functional hemichannel formation in the human but not in the rat Cx26 is combined with a different pattern of connexin expression. Rather than the abundant Cx26 expression in the developing and mature rodent epidermis (50) , human Cx26 is expressed at early stages of fetal epidermal development, but it gradually disappears and is undetectable during the stage of interfollicular keratinization and in the normal adult interfollicular epidermis (24 , 26) . These data may reflect the close relation between the functionality of the hemichannel and the function of Cx26 in the human epidermis. The expression of Cx26 was up-regulated in the palmoplantar epidermis and in tape-stripped skin, as well as in hyperproliferative disorders (25 , 49) , suggesting that Cx26 appears to maintain keratinocyte homeostasis during rapid growth and differentiation.

In summary, we show that in addition to contributing to gap junction-mediated intercellular communication, human but not rat Cx26 can form functional hemichannels capable of fulfilling a physiologically relevant role. Such species specificity should be taken into account when elucidating the normal and abnormal function of Cx26 in humans, particularly with respect to auditory function and in the epidermis. Moreover, mutant hemichannels with altered voltage gating and permeation properties may be implicated in the pathogenesis of hereditary deafness and ectodermal disorders associated with mutations in the Cx26 gene (16 , 51) .


   ACKNOWLEDGMENTS
 
We thank Rosa Barquero for outstanding technical assistance and Dr. Luis Escribano (Hematology Service of "Ramón y Cajal" Hospital) for his assistance with the FACS experiments. This work was funded by grants from the "Fondo de Investigaciones Sanitarias" 99/0203 and "Ministerio de Educación y Ciencia" SAF2005–03414 (to L.C.B.) and the Instituto de Salud "Carlos III" (Red TAU-G03/203). J.M.G-H is a researcher supported by the "Ramón y Cajal" Program (Ministerio de Educación y Ciencia).

Received for publication January 25, 2006. Accepted for publication June 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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