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Functional analysis of hemichannels and gap-junctional channels formed by connexins 43 and 46.
Hoang QV
,
Qian H
,
Ripps H
.
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PURPOSE: The gap junctions (GJs) mediating direct cell-cell interaction are formed by clusters of membrane-spanning proteins known as connexins (Cxs). These channels play a key role in signal transmission, and their permeability, time-, and voltage-dependence are governed by the properties of the specific Cxs forming the gap junctions. Retinal pigment epithelium (RPE) cells express Cx43 and Cx46. Here, we employed a heterologous expression system to explore the functional properties of the hemichannels and GJs that could be formed by different combinations of these Cxs. Specifically, we examined the response kinetics of GJs formed by pairing cells expressing Cx43 or Cx46, or those expressing both, i.e., designated as Cx43*Cx46.
METHODS: The Xenopus oocyte expression system and a two-electrode voltage clamp technique were used to study the properties of hemichannels and GJs formed in oocytes transfected with Cx43 and/or Cx46 mRNA.
RESULTS: Depolarizing voltages activated hemicurrents of similar amplitude from single oocytes transfected with Cx46 or Cx43*Cx46, but not in oocytes expressing Cx43 alone. Incorporating Cx43 with Cx46 altered the gating charge, but not the voltage sensitivity of the hemichannels. In addition, Cx43*Cx46 hemichannel currents exhibited faster activation kinetics than homomeric Cx46 hemichannels. Both homotypic GJs formed by Cx43 and Cx46, and heteromeric Cx43*Cx46 GJs exhibited large junctional conductances with amplitudes of 6.5+/-3.0 microS (Cx43), 8.9+/-3.4 microS (Cx46), and 8.5+/-1.8 microS (Cx43*46); a significantly lower conductance (1.8+/-0.7 microS) was observed for heterotypic GJs formed by Cx43 and Cx46. There were also differences in their gating kinetics. Whereas the kinetics of homotypic Cx46 could be described by a single exponential function (tau=0.91 s), double exponential functions were required for homotypic Cx43 (tau(1)=0.24, tau(2)=3.4 s), heterotypic Cx43/Cx46 (tau(1)=0.29, tau(2)=3.6 s), and heteromeric Cx43*Cx46/Cx43*Cx46 (tau(1)=1.2, tau(2)=8.1 s) junctions.
CONCLUSIONS: The failure of oocytes expressing Cx43 to exhibit hemichannel activity is an intrinsic membrane property of this Cx, and cannot be attributed to a lack of expression; western blot analysis showed clearly that Cx43 was expressed in oocytes in which it was injected. Our results provide further evidence that Cx43 and Cx46 form both heterotypic and heteromeric channels when co-expressed, an indication that various combinations of Cxs may participate in gap-junctional communication between RPE cells.
Figure 1. Membrane currents recorded from single cells in response to depolarizing voltage steps from a holding potential of −20 mV, and stepped in 10 mV increments from −10 mV to +60 mV. The oocytes were injected with 46 nl of an aqueous solution containing an antisense nucleotide to the endogenous Cx (Cx38) normally expressed by Xenopus oocytes, either alone (A) or along with (B) 13 ng of the cRNA encoding Cx43, (C) a similar concentration of Cx46, and (D) a mixture containing 13 ng each of Cx43 and Cx46. Note that the outward currents typical of hemichannel activity were absent from oocytes receiving either the antisense oligomer or Cx43.
Figure 2. Current-voltage relationships (averaged data) for cells expressing Cx46 (n=9) and the combined cRNA derived from the mixture of Cx43 and Cx46 (n=11). The curves for the antisense (control, n=10) and Cx43 (n=12) injected cells remained at baseline over the entire voltage range.
Figure 3. The voltage activation profile examined by tail current analysis in hemichannels from representative cells expressing (A) Cx46 or (B) the combined cRNA derived from the mixture of Cx43 and Cx46 (Cx43•Cx46). The amplitude of the tail current was derived by extrapolating back to the end of the 10 s voltage step. 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.
Figure 4. The kinetics of hemichannel activation determined from the response to a +40 mV voltage step in representative cells expressing (A) Cx46 or (B) the combined cRNA derived from the mixture of Cx43 and Cx46 (Cx43•Cx46). For both types of hemichannels, the activation time course was best fit with a two-exponential function. The mean activation time constants (τ) are shown for Cx46 (n=9) and Cx43•46 (n=12). Note that Cx43•46 hemichannel currents exhibit faster activation kinetics than homomeric Cx46 hemichannels.
Figure 5. Junctional current (Ij) data from paired oocytes. The left column 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 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).
Figure 6. The kinetics of the voltage-dependent closure of gap junctional channels for homotypic (A, B), heterotypic (C), and heteromeric (D) pairs of RPE Cxs expressed in Xenopus oocytes. For homotypic pairs of Cx43/Cx43 (A), the junctional current was best fit with a second-order exponential decay, whereas homotypic Cx46 (B) could be described by a single exponential function with averaged time constants shown above (n=5 and n=3, respectively). Gating kinetics for the heterotypic Cx43/Cx46 gap junctional channel (C) also required two exponential components (n=6). Heteromeric gap junctions (Cx43•46/Cx43•46) exhibited significantly slower gating kinetics, requiring a second-order exponential function, with the time constants shown in D, above (n=10).
Figure 7. Western blot analysis of Cxs expressed in Xenopus oocytes. Lanes 1, 3, 5: uninjected oocytes to serve as non-expressing control. Lanes 2 and 6: oocytes co-expressing Cx43 and Cx46 (Cx43•46). Lane 4: oocytes expressing Cx43 alone. Lanes 1–6 were probed with anti-Cx43 antibody, whereas lanes 5 and 6 were probed with anti-Cx46 antibody.
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