Functional analysis of hemichannels and gap-junctional channels formed by connexins 43 and 46

Ethics statement

All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23), revised in 1996, and adhered to the guidelines for the care and use of laboratory animals formulated by the Association for Research in Ophthalmology and Visual Sciences (ARVO). All procedures were approved by the Animal Care Committee of the University of Illinois at the Chicago College of Medicine. Oocytes of the South African clawed toad Xenopus laevis are widely used as a system for expression and characterization of exogenous membrane channels. The cells provide a convenient test environment, are large and easily penetrated for microinjection and recording, and have been used extensively for heterologous expression of ion channels, neurotransmitter receptors, transporters, and other membrane proteins.

Oocyte preparation

The procedures for oocyte preparation and Cx expression were described previously [19]. Briefly, ovarian lobes were removed under surgical anesthesia (0.02% 3-aminobenzoic acid ethyl ester, Tricaine) from gravid Xenopus laevis females (Xenopus I, Dexter, MI), and incubated with constant agitation for 2 h in a calcium-free modified Barth’s (MB) solution containing collagenase (2.5 mg/ml), gently triturated, and rinsed twice in Ca²⁺-free MB without collagenase. Defolliculated stage V-VI oocytes were selected and repeatedly rinsed in MB solution containing (in mM): NaCl (88), KCl (1), NaHCO₃ (2.4), HEPES (N-2-hydroxyethylpiperazine-N’-2-ethaneesulfonic acid) (15), Ca(NO₃)₂ (0.33), CaCl₂ (0.41), and MgSO₄ (0.82); 10 mg/l gentamycin (Gibco/BRL) was added, and the solution was titrated with NaOH to pH 7.6.

Plasmids containing the coding sequences of mouse Cx43 and mouse Cx46, generously provided by Dr. Thomas White (SUNY, Stony Brook, NY), were subcloned into the BamH1 site of the pCS2+ expression vector. The constructs were linearized with the NotI restriction endonuclease, and capped mRNAs were transcribed in vitro with SP6 RNA polymerase using the mMessage mMachine (Ambion Inc., Austin, TX). The oocytes were maintained in MB at 15 °C for 2 h to two days before injection of cRNAs into the vegetal poles of the oocytes using a Nanoject Injector (Drummond Scientific Co., Broomall, PA). The injection pipettes were drawn on a vertical electrode puller (Narishige PB-7, Tokyo, Japan), and broken under microscopic observation to a tip opening of 10–15 μm. Oocytes were injected with 46 nl of an aqueous solution containing 13 ng/cell of Cx43 and/or 13 ng/cell of Cx46, together with 10 ng/cell of an antisense oligomer to nucleotides 128–151 of the coding region of the endogenously-expressed Cx38. For control experiments, oocytes were injected only with a solution containing 10 ng/cell of the antisense oligomer.

Electrophysiology

Nonjunctional (hemichannel) currents were recorded ~2 days after transfection from individual oocytes mounted on a 0.5 mm nylon mesh in a specially constructed lucite chamber (volume=0.7 ml) using a two-electrode voltage clamp [20]. Cells were clamped at −20 mV, and whole cell currents were recorded in response to depolarizing voltage steps (−10 to +60 mV at 10 mV intervals) imposed for a duration of 10 s. Current values measured at the end of the pulse were plotted against membrane potential. In experiments designed to determine gap junctional conductances, the vitelline envelopes of the oocytes were removed in a hypertonic solution one day after transfection, and the cells manually paired with vegetal poles apposed in Petri dishes containing MB supplemented with 5% Ficoll. For both types of experiments, current and voltage electrodes were pulled to resistances of 0.7–1.5 MΩ when filled with an internal solution of 3 M KCl, 10 mM EGTA, and 10 mM HEPES, at pH 7.4. The external solution was MB with 5% Ficoll.

Voltage gating and related properties of the intercellular channels between oocytes were measured ~two days after transfection, and one day after oocyte pairing using a dual-cell voltage-clamp technique. To quantitate junctional conductance (G_j), the two cells of a pair were clamped at −40 mV, close to their initial resting potential, and transjunctional current measurements were obtained in response to 20 mV depolarizing steps applied alternately to each of the cells. Under these conditions, the current applied by the clamp to the cell not stepped is equal in amplitude, but opposite in sign to the junctional current (I_j). Junctional conductance was then calculated by dividing the junctional current by the transjunctional potential. To determine the voltage-gating properties of the intercellular channels, transjunctional potentials of opposite polarity were generated by hyperpolarizing or depolarizing one cell in 20 mV steps (over a range of ±120 mV), while clamping the second cell at −40 mV. After each voltage step, steady-state currents (I_jss) were measured 10 s after the onset of the voltage pulse. Steady-state conductances (G_ss) were normalized to their value at ±20 mV, and plotted against the transjunctional potential. Voltage clamping of paired oocytes was performed using two GeneClamp 500B amplifiers controlled by pClamp 8 software through a Digidata 1322A interface (Axon Instruments, Molecular Devices, Sunnyvale, CA). Hemichannel currents and GJIC currents were analyzed using the pClamp 8 software suite (Axon Instruments).

Data describing the macroscopic junctional conductance as a function of transjunctional voltage were analyzed using Microcal Origin 5.0 software (Microcal Software Inc., Northampton, MA) and fit to a Boltzmann relation [21] of the form:

G_jss={(G_jmax – G_jmin)/(1 + exp[A (V_j – V_o)])} + G_jmin (1)

where G_jss is the steady-state junctional conductance, G_jmax (normalized to unity) is the maximum conductance, G_jmin is the residual conductance at large values of junctional voltage (V_j), and V_o is the transjunctional voltage at which G_jss=(G_jmax – G_jmin)/2. The constant A (=nq/kT) represents the voltage sensitivity in terms of gating charge as the equivalent number (n) of electron charges (q) move through the membrane, k is the Boltzmann constant, and T is the absolute temperature. The time constants (τ) of voltage-dependent transitions of junctional conductance were calculated using data fitting functions in Microcal Origin.

Western-blot analysis

Oocytes were harvested two days after transfection with Cx mRNA. Non-expressing oocytes served as controls. The membrane protein fraction of the oocytes was prepared using the method described previously [22]. Next, 50 μg of total membrane proteins were loaded in each lane and separated on SDS–PAGE. After transfer to a polyvinylidene difluoride membrane, the blot was probed with primary antibody (1:500 dilutions; Invitrogen) overnight at 4 °C, followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibody. The signals on the blot were detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL).

Statistical analysis

Data analysis tools in Microsoft Excel were used to calculate statistical significance (two-tail t-test analysis).

Hemichannel currents

Figure 1 illustrates examples of membrane currents elicited by voltage pulses from individual Xenopus oocytes injected with antisense Cx38 oligonucleotide alone or expressing Cx43, Cx46, or Cx43 with Cx46 (Cx43•46). In oocytes injected with the antisense Cx38 nucleotide, minimal membrane currents were observed in response to voltage steps from −10 mV to +60 mV (Figure 1A). This finding is similar to the results obtained by Ebihara [23], who reported that large hemichannel currents (>100 nA at +30 mV) are elicited from oocytes not injected with the antisense oligomer, whereas after injection the current is close to zero. As shown in Figure 1A and Figure 2, the antisense oligomer had fully blocked any hemichannel activity that might be attributed to Cx38. Interestingly, the recordings obtained from oocytes expressing Cx43 did not differ from those with the antisense nucleotide, i.e., there was no evidence of significant hemichannel activity (Figure 1B). This is consistent with observations reported in several studies showing that Cx43 hemichannels have a very low open probability under normal conditions in those tissues in which they are expressed [24]. However, they can be opened, e.g., in cardiac myocytes, in response to mechanical, osmotic, and oxidative stress or changes in extracellular or intracellular Ca²⁺, whereupon they release small ions and molecules with signaling potential such as ATP [25]. On the other hand, when expressed in oocytes, the hemichannel conductance is so low as to be undetectable (Figure 1B and Figure 2), a finding first reported by White et al. [26]. In contrast, voltage-activated outward hemicurrents were obtained from cells transfected with Cx46 in response to depolarizing voltage steps ≥+10 mV (Figure 1C), and similar current responses were observed in oocytes co-expressing Cx46 with Cx43 (Figure 1D).

Figure 2 illustrates the mean voltage-activated currents, averaged from several cells, for the various Cx hemichannel configurations. Note that responses of similar magnitude were obtained from oocytes expressing Cx46 and Cx43•46 over the entire range of voltages tested, and in response to a +60 mV voltage pulse, the hemichannel currents were 13.7±1.3 µA for Cx46 (mean±SEM, n=9), and 12.4±1.3 µA for Cx43•Cx46 (mean±SEM, n=11). In contrast, oocytes expressing Cx43 (n=12) or the antisense oligo (n=10) gave extremely small inward currents at this voltage that did not differ significantly from baseline. In a few instances, larger inward currents were seen, probably due to the endogenous sodium channels present in these cells [27].

The voltage activation profile of the hemichannels expressed by Cx46 and Cx43•46 was examined using tail current analysis. The amplitude of the tail current was derived by extrapolating back to the end of the 10 s voltage step using data fitting functions in the pClamp 8 software suite. Examples of the voltage activation profile for hemichannels formed in oocyte membranes by Cx46 and Cx43•46 are shown in Figure 3; the continuous lines represent curves fit with the Boltzmann equation. Although of similar shape, the curve describing the hemicurrent recorded from oocytes expressing Cx43•46 has a steeper slope, which reflects the difference in gating charge (n) in the Boltzmann equation.

Table 1 presents the averaged values for the parameters used in fitting the Boltzmann equation. Notably, the gating charge (n) for Cx46 was found to be 1.4±0.05 (mean±SEM, n=9) and it was 1.6±0.07 (mean±SEM, n=11) for Cx43•46; the difference is statistically significant with a p-value of 0.01 (two-tailed t-test). These results indicate that incorporating Cx43 with Cx46 alters the voltage sensitivity of the hemichannels expressed in the oocyte membrane. On the other hand, values of V_o, the membrane voltage at which half of the channels are activated, are similar for cells expressing Cx46 or Cx43•46, i.e., 39.3 mV for Cx46 and 39.5 mV for Cx43•46 (Table 1).

The kinetics of hemichannel activation were examined for voltage steps to +40 mV, a potential close to V_o for both Cx46 and Cx43•46. For both hemichannels, the activation time course was best fit with a two-exponential function (examples shown in Figure 4). The mean activation time constants (τ) were 0.89±0.07 s and 5.9±1.0 s (n=9) for oocytes expressing Cx46, and 0.69±0.04 s and 4.3±0.5 s (n=12) for cells expressing Cx43•46. In other words, Cx43•46 hemichannel currents exhibit faster activation kinetics than homomeric Cx46 hemichannels. These differences are statistically significant for the fast phase of the time constant (p=0.017, two-tailed t-test), but not for the slow phase (p=0.14). In addition, the proportion of fast and slow components in the hemichannel currents is also statistically insignificant for cells expressing Cx46 alone and those co-expressing Cx43 and Cx46 (p=0.4).

Gap junctional conductance

Figure 5 illustrates junctional current (I_j) data from paired oocytes. The left column in Figure 5 shows examples of current traces recorded from homotypic pairs (Cx43/Cx43 and Cx46/Cx46), a heterotypic pair (Cx43/Cx46), and a heteromeric pair (Cx43•46/Cx43•46). The right column in Figure 5 plots normalized steady-state junctional conductances (filled circles) measured at the end of 10-s-long voltage steps, and fit to the Boltzmann equation (curved lines). The corresponding Boltzmann parameters for positive and negative values of V_j, including the gating charge n, are given in Table 2.

For both homotypic pairs formed in oocytes, large junctional conductances (G_j) were observed, with values of 6.5±3.0 μS (Cx43, n=5) and 8.9±3.4 μS (Cx46, n=3; Table 3). On the other hand, the junctional conductance of the heterotypic channels formed by Cx43 and Cx46 was significantly less (1.8±0.7 μS, n=6). In addition, a large asymmetry can be seen for heterotypic junctional channels, with voltage sensitivities of 92 mV and 46 mV, and G_jmin of 0.40 and 0.23 at +V_j and –V_j, respectively. A large junctional conductance was also observed in paired oocytes expressing both Cx43 and Cx46 (8.5±1.8 μS, n=10). The pattern of voltage sensitivity for the junctional current in these cells is more similar to homotypic pairs than the asymmetry displayed by heterotypic pairs.

The gating kinetics of the gap junctional channels were derived from the responses to –120 mV voltage pulses. Figure 6 shows examples of junctional current traces recorded from each of the junctional pairs investigated in this study. For homotypic pairs of Cx43/Cx43, the junctional current was best fit with a second-order exponential decay, with averaged time constants of 0.24±0.02 s and 3.4±0.3 s (n=5). Interestingly, the kinetics of the voltage-dependent closure of gap junctional channels formed by homotypic Cx46 could be described by a single exponential function with a mean time constant of 0.91±0.02 s (n=3). As the heterotypic Cx43/Cx46 gap junctional channel exhibits a large asymmetry in voltage sensitivity, we analyzed the gating kinetics at both +V_j and –V_j when Cx46 cells were held at constant voltage. For both the positive and negative junctional potentials, the gating kinetics for the heterotypic Cx43/Cx46 gap junctional channel also required two exponential components with values of 0.29±0.03 s and 3.6±0.3 s (n=6) for –V_j, and 0.81±0.08 s and 5.1±0.9 s for +V_j. On the other hand, the heteromeric gap junction formed by co-expressing Cx43 and Cx46 (i.e., Cx43•46/Cx43•46) exhibited significantly slower gating kinetics, requiring a second-order exponential function with time constants of 1.2±0.3 s and 8.1±2.3 s (n=10).

Connexin expression in oocyte membranes

The expression of Cx subunits in oocyte membranes was probed using western blot analysis. As shown in Figure 7, no Cx specific signals were detected from samples prepared from non-expressing oocytes (lanes 1, 3, 5). For oocytes co-expressing Cx43 and Cx46 (Cx43•46), a Cx43 specific band was detected with a molecular mass of about 43 kDa when probed with anti-Cx43 antibody (lane 2), and a Cx46 band was detected with a molecular mass of about 46 kDa when probed with anti-Cx46 antibody (lane 6). For oocytes expressing Cx43 alone, a band at the appropriate molecular mass of ~43 kDa was also detected in the membrane preparation of the sample (lane 4). These results indicate that the inability to detect hemichannel currents in oocytes expressing Cx43 is not due to a lack of protein synthesis of the subunit in the membrane, but most likely reflects the unique gating property of the Cx43 connexon.