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Life (Basel)
2023 Jan 31;132:. doi: 10.3390/life13020390.
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Divergence between Hemichannel and Gap Junction Permeabilities of Connexin 30 and 26.
Xu J
,
Nicholson BJ
.
???displayArticle.abstract??? Cx30 has been proposed to play physiological functions in the kidney and cochlea, and this has often been associated with its hemichannel role (deafness mutants frequently affecting hemichannels more than gap junctions), implicated in ATP release. Here, we used heterologous expression systems (Xenopus oocytes and N2A cells) to describe the properties of Cx30 hemichannels, with the objective of better understanding their physiological functions. As previously observed, Cx30 hemichannels gated in response to transmembrane voltage (V0) and extracellular [Ca2+] (pK[Ca2+] of 1.9 μM in the absence of Mg++). They show minimal charge selectivity with respect to small ions (ratio of Na+: K+: Cl- of 1: 0.4: 0.6) and an MW cut-off for Alexa Dyes between 643 (Alex 488) and 820 Da (Alexa 594). However, while cations follow the expected drop in conductance with size (Na+ to TEA+ is 1: 0.3), anions showed an increase, with a ratio of Cl- to gluconate conductance of 1:1.4, suggesting favorable interactions between larger anions and the pore. This was further explored by comparing the permeabilities of both hemichannels and gap junctions to the natural anion (ATP), the release of which has been implicated in Ca++ signaling through hemichannels. We extended this analysis to two closely related connexins co-expressed in the cochlear, Cx26 and Cx30. Cx30 and 26 hemichannels displayed similar permeabilities to ATP, but surprisingly Cx26 gap junctions were six times more permeable than their hemichannels and four times more permeable than Cx30 gap junctions. This suggests a significant physiological difference in the functions of Cx26 and Cx30 gap junctions in organs where they are co-expressed, at least with regard to the distribution of energy resources of the cells. It also demonstrates that the permeability characteristics of hemichannels can significantly diverge from that of their gap junctions for some connexins but not others.
Figure 1. Immunostaining for Cx30 of sections from control oocytes without RNA injection (A) and hCx30WT-expressing oocytes (B). Regions of the vegetal pole are shown. Scale bars represent 100 µm.
Figure 2. Cx30 forms hemichannels in Xenopus oocytes and N2A cells: (A–D): Oocyte expression system; transmembrane currents in response to increasing voltage steps of both polarities in oocytes bathed in Ca2+-free ND96 solution injected with antisense oligo alone (A) or in conjunction with hCx30 RNA (B). Initial current amplitudes plotted against the voltage of Cx30- and antisense-oligo-injected oocytes are linear (C), while steady-state conductances plotted against the voltage of Cx30-expressing oocytes (with antisense oligo subtracted) show a partial closure at membrane potentials below −20 mV (Boltzman fit shown in red—V0 ~ −35 mV) (D). (E–H): N2A expression system; transmembrane current in response to the increasing voltage steps of both polarities from control (E) and hCx30-expressing N2A cells (F) bathed in a Ca2+-free solution. Initial current amplitude plotted against voltage of transmembrane recordings of control and hCx30-expressing N2A cells are linear, with a slight increase at −80 mV in Cx30-expressing cells (G). Steady-state membrane conductance plotted against the voltage of Cx30-expressing N2A cells (conductance from control cells subtracted) showed a decay at membrane potentials below −60 mV (Boltzman fit shown in red) (H).
Figure 3. Cx30 hemichannels sensitivity to extracellular [Ca2+]: (A) If Ca++ is removed from the media, then Cx30-expressing oocytes show a drop in membrane potential to −10 mV compared with −30 mV in the absence of Cx30 expression, while in the presence of normal extracellular Ca++, no significant difference in membrane potential was evident. (B) By chelating extracellular Ca2+ with EGTA to a series of concentrations, normalized membrane conductance could be plotted against the log of [Ca2+]o concentration to measure the Ca++ sensitivity of Cx30 hemichannels (Kd of ~ 10 µM). ** p < 0.01; *** p < 0.001, based on a two-tailed t-test.
Figure 4. I–V relations of Cx30 hemichannels in 24, 120 and 240 mM extracellular NaCl. Current traces of Cx30 hemichannels were recorded in 24 mM (blue), 120 mM (red) and 240 mM (black) extracellular NaCl. Calcium in all the solutions is chelated by EGTA to 10 nM. Note that the intersection point of the currents is in the first quadrant and that reversal potentials shift to positive values with increasing NaCl concentration, allowing calculations of relative Na+, K+ and Cl− conductances by the GHK equation.
Figure 5. Permeability of Cx30 hemichannel to Dyes and ATP. (A) Three Alexa dyes of increasing size (350 (MW 350), 488 (MW 570) and 594 (MW 760)) were injected into oocytes previously injected with antisense oligo to endogenous XeCx38 alone (white) or co-injected with cRNA for Cx26 (grey) or Cx30 (black). Fluorescence intensity was released following incubation in Ca2+-free KCl solution, which was measured at 60 min. (B) Precisely 32 nL of 1.25 mCi/mL 35S-labeled ATP-γ-S was injected into oocytes as described in (A). Radiation released following incubation in Ca2+-free KCl solution was measured at 60 min. Data are means ± SEM; n = 5; ** p < 0.01; *** p < 0.001, based on a two-tailed t-test.
Figure 6. Comparison of Cx26 and Cx30 hemichannel and gap junction channel permeability to ATP-γ-S. (A) Linear fit of the ratio of released ATP-γ-S to the medium (M) to ATP-γ-S retained in the injected oocyte (O) against electrical conductance, measured by a two-electrode voltage clamp prior to injection. The average value of released ATP-γ-S and electrical conductance from control oocytes (not expressing connexins) is subtracted from each measurement. (B) Linear fit of ATP-γ-S transferred from an injected donor (D) to acceptor (A) oocyte through gap junction channels (presented as A/D ratio) against transjunctional conductance immediately measured prior to ATP injection into the donor cell. The average value of ATP-γ-S transfer and electrical conductance from control oocytes (not expressing connexins) is subtracted from each measurement. (C,D) Since the recordings are collected under identical conditions in terms of ATP injection and time of transfer, the plots in A and B can be combined on the same axes to provide a direct comparison of hemichannel and gap junction channel permeabilities for Cx30 (C) and Cx26 (D). In all cases, ATP injection and collection were performed as described in Figure 4. For the gap junction assays, the acceptor and donor oocytes are separated and processed for counting after 1 h.
Figure 7. Model of the physiological effects of differential permeabilities of Cx26 and 30 hemichannels and gap junctions.
Figure S1: Superimposed intercellular current traces from dual oocyte voltage clamp of paired oocytes injected with Cx26 wtRNA.
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