1Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794; and 2Department of Zoology, University of Manitoba, Winnipeg, Canada R3T 2N2
Submitted 6 May 2004 ; accepted in final form 23 July 2004
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ABSTRACT |
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connexin; gating; retina
In addition to forming gap junction channels between two communicating cells, many connexins can also form voltage-activated hemichannels within single plasma membranes (1, 2, 7, 14, 46, 49, 51, 55). Analysis of unopposed hemichannels in functional expression systems provides a unique opportunity to compare hemichannel properties with those of intact gap junction channels between coupled cells (47). A detailed knowledge of hemichannel conductance and gating is also a prerequisite for evaluating whether hemichannels might play any significant physiological roles in vivo (3, 11, 24, 52).
In situ hemichannel activity was first documented in horizontal cells isolated from fish retina (9, 28). To date, at least seven connexins have been identified in the retinas of several species of fish (8, 25, 29, 35, 36, 54, 59). Only two of these, Cx35 and Cx52.6, have been shown to be capable of forming active hemichannels in functional expression systems (32, 55, 56, 59), although neither has been characterized at the single channel level. This is potentially significant, because hemichannel activity has been implicated as an important component of feedback inhibition between cones and horizontal cells in the outer plexiform layer of the fish retina (20, 25, 40). It also has been suggested that hemichannels expressed in other retinal cell types may also affect synaptic transmission (59). In the perch and zebrafish retina, Cx35 has been immunolocalized to the inner and outer plexiform layers (29, 36), suggesting that it may be one candidate connexin to mediate hemichannel effects on synaptic transmission.
Full evaluation of the potential roles for zebrafish Cx35 in hemichannel-mediated inhibition in vivo first requires a complete documentation of hemichannel conductance and gating properties in vitro. Toward this goal, we have shown that Xenopus oocytes injected with Cx35 cRNA and N2A cells transfected with Cx35 cDNA develop large, outward whole cell currents on depolarization. Using cell-attached and excised patch configurations, we further demonstrated that both the unitary conductance (hc) and gating of Cx35 hemichannels exhibited a pronounced dependence on membrane voltage (Vm).
hc increased with relative hyperpolarization and decreased on depolarization, whereas channels gated closed with negative Vm and opened with positive Vm. The ability to document the conductance and gating of Cx35 hemichannels will help to clarify whether their functional properties are consistent with current models of hemichannel feedback inhibition in the retina.
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MATERIALS AND METHODS |
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Oocyte preparation. Stage V and VI oocytes were isolated from Xenopus laevis (Nasco, Fort Atkinson, WI), defolliculated by collagenase digestion, and cultured in modified Barth's (MB) medium. Cells were injected with a total volume of 40 nl of either an antisense oligonucleotide (3 ng/cell), to suppress endogenous Xenopus Cx38, or a mixture of antisense plus Cx35 cRNA (40 ng/cell), using a Nanoject II Auto/Oocyte injector (Drummond, Broomall, PA). After overnight incubation, oocytes were immersed for a few minutes in hypertonic solution to strip the vitelline envelope, transferred to petri dishes containing MB medium, and manually paired with the vegetal poles apposed for gap junction channel analysis. For hemichannel analysis, single oocytes were devitellinized and processed as described below. Electrophysiological recordings were made 48 h after cRNA injection.
Whole cell electrical recordings in Xenopus oocytes.
The functional properties of hemichannels and gap junction channels were assessed by dual voltage clamp (43). Current and voltage electrodes (1.2-mm diameter; Glass Company of America, Millville, NJ) were pulled to a resistance of 12 M with a horizontal puller (Narishige, Tokyo, Japan) and filled with 3 M KCl, 10 mM EGTA, and 10 mM HEPES, pH 7.4. Voltage clamping of oocyte pairs was performed using two GeneClamp 500 amplifiers (Axon Instruments, Foster City, CA) controlled by a PC-compatible computer through a Digidata 1320A interface (Axon Instruments). pCLAMP 8.0 software (Axon Instruments) was used to program stimulus and data collection paradigms. Current outputs were filtered at 50 Hz, and the sampling interval was 10 ms. To determine voltage-gating properties of gap junction channels, transjunctional potentials (Vj) of opposite polarity were generated by hyperpolarizing or depolarizing one cell for 3 s in 20-mV steps (over a range of ±120 mV) while the second cell was clamped at 40 mV. Currents were measured at the end of the voltage pulse, at which time they approached steady state (Ijss), and the macroscopic conductance (gjss) was calculated by dividing Ijss by Vj. gjss was then normalized to the values determined at ±20 mV and plotted against Vj. To characterize hemichannels, single oocytes were assessed with a two-electrode voltage-clamp procedure (14). Cells were initially clamped at 40 mV. Depolarizing voltage steps (20 to +80 mV at 20-mV intervals) were imposed for 5 s, and whole cell currents were recorded. To test the calcium dependence of Cx35 hemichannel currents, MB medium supplemented with 2 mM CaCl2 was used. Mean current values were measured at the end of the pulse and plotted against the clamped membrane potential (Vm).
Cells and culture conditions.
Experiments were carried out on N2A cells transiently transfected (Lipofectin; Invitrogen Life Technologies) with cDNA coding for zebrafish Cx35. N2A cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. The cells were cultured at 37°C in a CO2 incubator (5% CO2-95% ambient air). To perform experiments, the cells were harvested and seeded onto sterile glass coverslips placed in multiwell culture dishes (104 cells/cm2). Electrophysiological experiments were carried out on cells cultured for 13 days after transfection. Transfected cells were selected for electrophysiological analysis by GFP expression.
Solutions and pipettes.
During patch-clamp experiments, oocytes were superfused with bath solution containing (in mM) 120 potassium aspartate, 10 NaCl, 2 CaCl2, 5 HEPES (pH 7.4), and 5 glucose, with 2 mM CsCl, BaCl2, and tetraethylammonium chloride included. For the Ca2+-free (0 Ca2+) bath solution, CaCl2 was omitted. The patch pipettes were filled with solution containing (in mM) 120 potassium aspartate, 10 NaCl, 3 MgATP, 5 HEPES (pH 7.2), and 10 EGTA (pCa 8), filtered through 0.22-µm pores. For whole cell recording from N2A cells, potassium aspartate in the superfusate was replaced with an equal molar concentration of NaCl.
Electrical measurements.
Glass coverslips with adherent cells were transferred to an experimental chamber perfused with bath solution at room temperature (22°C). The chamber was mounted on the stage of an inverted microscope (Olympus IMT2). Patch pipettes were pulled from glass capillaries (code 7052; A-M Systems) with a horizontal puller (Sutter Instruments). When filled, the resistance of the pipettes measured 12 M
. Experiments were carried out on single cells using the whole cell voltage-clamp technique. A selected cell was attached to a patch pipette connected to a micromanipulator (WR-88; Narishige Scientific Instrument) and an amplifier (Axopatch 200). This method permitted control of the membrane potential, Vm, and allowed measurement of the associated membrane current, Im. Dual-whole cell patch clamp was used in experiments with cell pairs, allowing the control of the membrane potential of both cells and measurement of junctional currents (33). Initially, the membrane potentials of cell 1 and cell 2 were clamped to the same value, V1 = V2. V2 was then stepped to a new value to establish a transjunctional voltage, Vj = V2 V1. Currents recorded from cell 2 represent the sum of two components, the junctional current, Ij, and the membrane current of cell 2, Im2; the current obtained from cell 1 corresponds to Ij alone.
Signal recording and analysis.
Voltage and current signals were recorded using patch-clamp amplifiers (Axopatch 200). The current signals were digitized with a 16-bit analog-to-digital converter (Digidata 1322A; Axon Instruments) and stored with a personal computer. Data acquisition and analysis were performed with custom-made software (26) and pCLAMP8 software (Axon Instruments). Leak currents were not subtracted from any of the records. Curve fitting and statistical analyses were performed using SigmaPlot and SigmaStat, respectively (Jandel Scientific). The time constants () of voltage-dependent activation of hemichannel currents were determined using the data-fitting functions in pCLAMP8.
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RESULTS |
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Dependence of single channel activity on membrane potential.
Records obtained from Cx35-expressing oocytes in the cell-detached mode with a few single operational hemichannels were also used to study channel kinetics. The analysis of longer records (10-s duration) allowed us to demonstrate that the gating was voltage dependent at the single channel level. Figure 4 shows the operation of two to three Cx35 hemichannels in a cell-detached patch during different Vm steps. At a Vm of 30 mV, the channels were frequently open, with a few transitions to the closed state (upward current deflections). Increasing Vm to 50 and 70 mV resulted in an increased gating of Cx35 hemichannels to the closed state. Thus hyperpolarizing voltage tends to reduce the time in the open state, consistent with the macroscopic records, in which ensembles of channels behave as outward rectifiers.
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Hemichannel activity in N2A cells transfected with Cx35. To compare the effects of expression system on Cx35 hemichannels and intact gap junction channels, we transfected N2A cells with Cx35 cDNA. Figure 6A illustrates the voltage protocol used to activate whole cell hemichannel currents in transfected cells. A family of Im traces was elicited by depolarizing (outward currents) and hyperpolarizing pulses (inward currents). The records were obtained from N2A cells superfused with Ca2+-free solution. After whole cell recording conditions were established, the Vm was clamped to 0 mV and the bipolar voltage pulses of 5-s duration were then delivered to alter Vm in 20-mV steps from ±10 to ±90 mV. No significant currents were recorded in untransfected control N2A cells (Fig. 6B). In Cx35-transfected cells, the associated membrane current (Im) increased with depolarization (Vm) and showed a voltage- and time-dependent activation, whereas hyperpolarization induced inward currents that deactivated with time (Fig. 6C). In Cx35-expressing N2A cells, a plot of Im vs. Vm (Fig. 6D) demonstrated the same hemichannel current-voltage relationship as recorded in oocytes. Thus Cx35-expressing N2A cells developed currents that activated slowly, with depolarizing voltage steps that were similar to those recorded from Xenopus oocytes (see Fig. 1), and the functional behavior of Cx35 hemichannels was not influenced by the type of cell in which they were expressed.
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DISCUSSION |
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We also have characterized single and multichannel activity using cell-attached and -detached patch-clamp methods and have found that Cx35 behaved similarly to the other documented hemichannels (46, 49, 51). Cx35 hemichannels are voltage gated and behave similarly to outward rectifiers as has proven to be the case for all the other hemichannels analyzed at the single channel level. This voltage-gating behavior is consistent with both fast voltage-dependent gating and the slower purported "loop gating" (6, 39, 46), with the latter being sensitive to extracellular Ca2+. In addition, Cx35 is able to form typical gap junction channels in both oocyte pairs and N2A cell pairs, as shown in Fig. 8. The macroscopic data obtained from oocyte cell pairs confirm those previously published (29). In combination with the data obtained from N2A cell pairs in this study, we have demonstrated that Cx35 gap junction channel voltage-dependent properties are not altered by the choice of cell type used as an expression system.
Zebrafish Cx35 is highly expressed in the brain and retina, and structural analysis of both the gene and the protein clearly demonstrate that Cx35 belongs to the Cx35/36 subgroup of orthologous connexins (29). Immunohistochemical analysis has previously localized zebrafish Cx35 in the retina in both the inner plexiform layer, a synaptic layer for ganglion cells and interneurons such as bipolar cells and amacrine cells, and the outer plexiform layer, where dendrites of horizontal cells and bipolar cells synapse with photoreceptors (29). This pattern is consistent with many other studies showing Cx35/36 labeling in the retinas of other species (16, 17, 21, 22, 30, 31, 36, 45, 58). Within the fish brain, Cx35 has been localized in the mixed chemical and electrical synapses of goldfish Mauthner neurons (37, 38). To date, all of these immunolocalizations are consistent with the formation of patent Cx35 gap junction channels that function as classic electrotonic synapses (60), although asymmetric Cx35/36 labeling has been reported in the cone pedicle (27), which could correspond to the presence of either heterotypic gap junction channels or hemichannels.
Recently, hemichannel activity was proposed as a common mechanism mediating feedback inhibition between cones and horizontal cells in the outer plexiform layer of the vertebrate retina (25, 25, 40). Although Cx35 was not specifically implicated in this instance, its abundance in the retina makes it worthwhile to consider whether its functional properties, or the properties of other well-characterized hemichannels, are consistent with the model of feedback inhibition proposed (20). At issue is whether or not hemichannels have physiological properties consistent with their proposed function in synaptic transmission. On the basis of the present work and previous characterization of other connexin hemichannels, we can first define the minimal necessary conditions for hemichannel function in vivo.
Assuming hemichannels are stable structures within the membrane in vivo, the first important parameter limiting their function then appears to be the concentration of extracellular Ca2+. Previous studies have established that hemichannels are gated closed in the presence of millimolar Ca2+ levels presumably via interaction with the alleged "loop gate" (14, 18, 46, 49). Under what conditions would the interstitial fluid surrounding a cell be depleted of Ca2+? One possibility is a restricted space that is small in volume, where the membrane surfaces facing the space would have the capability to remove Ca2+. At least two of these criteria could be met in the case of cone pedicles and horizontal cells in the retina (25). They appear to interact in a restricted space with a small volume, although there is no direct experimental evidence to confirm these assumptions. The third requirement, significant reduction of extracellular Ca2+, has yet to be shown.
A second critical parameter for the activation of hemichannels is a depolarization of membrane voltage. All hemichannels characterized to date, including zebrafish Cx35, activate upon depolarization and gate closed upon hyperpolarization (7, 14, 46, 49, 51, 55). In the retinal feedback inhibition model (20, 25, 40), hemichannel current flow is assumed to increase as the horizontal cells hyperpolarize. This is counterintuitive to every documented example of hemichannel voltage dependence. Thus two important criteria for hemichannel activation, removal of extracellular calcium and cell depolarization, are inconsistent with their playing a role in feedback inhibition between cones and horizontal cells as previously proposed.
Pharmacological evidence for a hemichannel role in inhibition was provided by the use of the gap junction blocker carbenoxolone, which blocked feedback inhibition, and this effect was attributed to the specific loss of hemichannel activity (25). This view was recently challenged by a study showing that application of carbenoxolone at the same concentrations (100 µM) to patch-clamped, isolated cone photoreceptors directly reduced the cone Ca2+ current (53). Thus carbenoxolone directly inhibited cone Ca2+ channels and synaptic transfer independently of horizontal cell input, and this alone could explain the reduction in signaling previously attributed to inhibition of horizontal cell hemichannels.
The demonstration that some connexins can form functional hemichannels in vitro is currently not sufficient to allow a mechanistic understanding of how they impact cellular function, especially in the absence of supporting data showing that specific ionic requirements and other special conditions necessary for hemichannel gating have been met in the target tissue. To date, there is no direct in vivo or in situ demonstration of hemichannel activity that unequivocally demonstrates involvement in any form of extracellular mediated communication, although this remains an exciting, if elusive, hypothesis (3, 11, 20, 24, 52). Future hypotheses of hemichannel relevance to organ physiology must first be based on a thorough understanding of specific hemichannel functional properties of the connexin(s) involved. For Cx35, the present study provides an initial foundation for future investigation of this growing area of research.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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