Two distinct heterotypic channels mediate gap junction coupling between astrocyte and oligodendrocyte connexins

Abstract

Genetic diseases demonstrate that the normal function of CNS myelin depends on connexin32 (Cx32) and Cx47, gap junction (GJ) proteins expressed by oligodendrocytes. GJs couple oligodendrocytes and astrocytes (O/A channels) as well as astrocytes themselves (A/A channels). Because astrocytes express different connexins (Cx30 and Cx43), O/A channels must be heterotypic, whereas A/A channels may be homotypic or heterotypic. Using electrophysiological and immunocytochemical approaches, we found that Cx47/Cx43 and Cx32/Cx30 efficiently formed functional channels, but other potential heterotypic O/A and A/A pairs did not. These results suggest that Cx30/Cx30 and Cx43/Cx43 channels mediate A/A coupling, and Cx47/Cx43 and Cx32/Cx30 channels mediate O/A coupling. Furthermore, Cx47/Cx43 and Cx32/Cx30 channels have distinct macroscopic and single-channel properties and different dye permeabilities. Finally, Cx47 mutants that cause Pelizaeus-Merzbacher-like disease do not efficiently form functional channels with Cx43, indicating that disrupted Cx47/Cx43 channels cause this disease.

Figure 1.

Cx32/Cx30 and Cx47/Cx43 form overlapping puncta. A–E, HeLa cells stably expressing Cx30, Cx32, Cx43, or Cx47 were transiently transfected to express DsRed (DsRed+) and mixed with DsRed− cells in a ratio of 1 to 20. After 24 h, cells were immunostained as indicated, and counterstained for DAPI. One of the two possible pairings for each combination is illustrated. The DsRed+ cell is pseudocolored blue in the first and third rows, and indicated by an asterisk in the fourth row. Note that examples of Cx32/Cx30 (A) and Cx47/Cx43 (B) mixtures have overlapping puncta (arrowheads), whereas Cx47/Cx30 (C), Cx43/Cx32 (D), and Cx30/Cx43 (E) do not. Scale bar: 10 μm. To better illustrate that the puncta are localized to the cell membrane, the DsRed signal is enhanced in these images in supplemental Figure 1 (available at www.jneurosci.org as supplemental material). F, Quantitative summary of three independent experiments. For each mixture, an asterisk denotes the DsRed+ cell. Each dot shows the number of overlapping puncta determined for 1 DsRed+ cell. In each column, the horizontal bar denotes the mean, the vertical bar represents the 95% confidence interval, and the total number of DsRed+ cells is shown in parentheses. Only mixtures of Cx32/Cx30 (30*/32 and 32*/30) and Cx47/Cx43 (43*/47 and 47*/43) have overlapping puncta, in contrast to the other mixtures. The discrepancy between the number of overlapping puncta for Cx30*/Cx32 relative to Cx32*/Cx30 is likely caused by lower expression of Cx32. A–F, The mixtures are designated as potential O/A, A/A, or O/O pairs.

Figure 2.

Cx32/Cx30 and Cx47/Cx43 form functional channels. Neuro2A cells were transiently transfected with pIRES2-EGFP or pIRES2-DsRed vectors containing Cx30, Cx32, Cx43, Cx47, or no insert [vector alone control (CTR)]. After 24 h, EGFP- and DsRed-expressing cells were mixed in a 1 to 1 ratio; pairs were assessed by dual whole-cell patch clamping 6–48 h later. For each combination listed on the x-axis, the mean and SE of the G_j and number of pairs tested is shown. Using a Kruskal–Wallis test followed by Dunn's multiple comparison test, only Cx32/Cx30 and Cx47/Cx43 channels have G_j that is significantly greater (*p < 0.001) than those of the corresponding control pairs (32/CTR and 30/CTR or 43/CTR and 47/CTR).

Figure 3.

Voltage gating of Cx32/Cx30 and Cx47/Cx43 channels. A, C, Representative macroscopic current traces for Cx32/Cx30 (A) and Cx47/Cx43 (C) channels. Both cells of a pair were voltage clamped at the same voltage, then cell 1 (expressing Cx30 or Cx43) was stepped in 20 mV increments from V_j = −100 to V_j = +100 mV, and I_js were recorded from cell 2 (expressing Cx32 or Cx47). Note that the polarity of I_j is opposite that of V_j. For Cx32/Cx30 channels, the rate of I_j decay increased with increasingly positive V_j. For the reverse polarity of V_j, I_j showed slight decay only at −100 mV. The Cx32/Cx30 channels show substantial instantaneous rectification, with at least 3.5-fold greater current at V_j = +100 mV than at −100 mV. For Cx47/Cx43 channels, the rate of current decay increases as the absolute value of V_j increases, with some asymmetry in the kinetics of decay. Traces were filtered at 200 Hz. Because all traces were normalized to a +20 mV prepulse, amplitudes of the unitary transitions shown in these figures may not accurately reflect the single-channel sizes of these channels. However, single-channel conductances calculated from the raw data for this and other similar experiments are in agreement with records such as those shown in Figure 4. B, D, Average normalized G_j–V_j relations for heterotypic Cx32/Cx30 and Cx47/Cx43 channels. The average normalized instantaneous (open triangles) and steady-state (filled squares) G_j at each V_j were calculated from the current traces such as those shown in A and C, as described in Materials and Methods. Note that rapid gating to closure may have led to underestimates of instantaneous G_j for Cx32/Cx30 channels when the Cx30 cell was stepped to positive voltages (B), and on both polarities with Cx47/Cx43 channels (D).

Figure 4.

Single-channel recordings of Cx32/Cx30 and Cx47/Cx43 channels. A, B, The single-channel conductances of Cx32/Cx30 (A) and Cx47/Cx43 (B) channels were determined by applying voltage ramps from +100 to −100 mV to cell 1 (expressing Cx30 or Cx43; bottom) of a poorly coupled pair, and measuring the I_j in cell 2 (expressing Cx32 or Cx47, top). The conductance of the Cx32/Cx30 channel (A) shows greater than fivefold rectification between +100 to −100 mV, measuring ∼126 or ∼24 pS when the Cx30 cell is stepped to +100 mV or −100 mV, respectively, which is greater than the predicted rectification from the steady-state G_j–V_j plot (∼3.5-fold in Fig. 3) (see Results). Consistent with the macroscopic steady-state G_j–V_j plot, voltage gating of the Cx32/Cx30 channel is very asymmetric, with channel closure apparent only when the Cx30-cell is pulsed to positive voltages. Examples of Cx32/Cx30 channel closure (arrows) and opening (arrowheads) are noted. The conductance of Cx47/Cx43 channels (B) shows minimal rectification, measuring ∼70 or ∼80 pS when the Cx43 cell is stepped to +100 mV or −100 mV, respectively. This corresponds to ∼15% instantaneous rectification over the interval, in agreement with the conclusion from the macroscopic G_j–V_j plot. Gating of the Cx43 hemichannel likely accounts for closures with negative V_j (down arrows) whereas gating of the Cx47 hemichannel likely accounts for closures when the polarity of V_j is reversed (up arrows), because both of these hemichannels have negative gating polarities. The fourth upward arrow denotes what is likely a rapid channel closure. Traces were filtered at 100 Hz (A) and 150 Hz (B).

Figure 5.

Cx32/Cx30 and Cx47/Cx43 channels have different permeabilities. Neuro2A cells were transiently transfected with pIRES2-EGFP or pIRES2-DsRed vectors containing Cx30, Cx32, Cx43, or Cx47. After 24 h, EGFP- and DsRed-expressing cells were mixed in a 1 to 1 ratio. A–D, One cell of each Cx32/Cx30 or Cx47/Cx43 pair (denoted by an asterisk in each leftmost panel) was patched with an electrode filled with the GJ permeant dye AF350 (3.3 mm; A, C) or LY (0.1%; B, D). For each set of images, the leftmost panels show phase images, the middle panels show epifluorescence images of the patched cell filling with dye, and the right panels show whether dye transferred to a neighboring cell at least 75 s after establishing whole-cell configuration. Cell pairs containing Cx32/Cx30 channels transferred AF350 (A) but not LY (B). Cell pairs containing Cx43/Cx47 channels are permeable to both AF350 and LY. G_j for each experiment were 19.8 ns (A), 12.7 nS (B), 20.1 nS (C), and 6.7 nS (D). AF350: MW, 350; −1 charge; Lucifer yellow: MW, 443; −2 charge.

Figure 6.

Cx47 mutants do not efficiently form heterotypic channels. A–C, HeLa cells stably expressing Cx47 mutants (P87S, Y269D, or M283T) or Cx43 were transiently transfected to express DsRed (DsRed+) and mixed with DsRed− cells in a ratio of 1 to 20. After 24 h, cells were immunostained as indicated and counterstained for DAPI. One of the two possible pairings for each combination is illustrated. The DsRed+ cell is pseudocolored blue in the first and third rows, and indicated by an asterisk in the fourth row. Note that only M283T/Cx43 (C) pairings have overlapping puncta at the border of the DsRed signal (arrowhead) similar to Cx47/Cx43 pairings (Fig. 1), whereas pairing cells expressing Cx43 with those expressing other mutants do not. D, Quantitative summary of three independent experiments such as illustrated in A–C. The asterisk denotes the DsRed+ cell. Each dot represents the number of overlapping puncta determined for 1 DsRed+ cell. In each column, the horizontal bar denotes the mean, the vertical bar represents the 95% confidence interval, and the total number of DsRed+ cells is shown in parentheses. Both pairings of M283T/Cx43 (43*/M283T and M283T*/43) have overlapping puncta similar to pairings of Cx47/Cx43 (43*/47 and 47*/43) (data from Fig. 1). E, Neuro2A cells were transiently transfected with a pIRES2-EGFP or a pIRES2-DsRed bicistronic expression vector that also contained Cx43, Cx47, P87S, Y269D, or M283T. After 24 h, different combinations of red and green cell pairs were generated by mixing the transfected cells at a 1 to 1 ratio, and assessed by dual whole-cell patch clamping 24–48 h later. For each combination listed on the x-axis, the mean and SE of the G_j and number of pairs tested is shown. Using Kruskal–Wallis test followed by Dunn's multiple comparison test, the pairs containing mutant Cx47 have a G_j significantly different from that of WT Cx47/Cx43 channels (data from Fig. 2) (p < 0.001, P87S/Cx43; p < 0.05, Y269D/Cx43; p < 0.01, M283T/Cx43), but not control pairs (43/CTR or 47/CTR)(data from Fig. 2). Scale bar: 10 μm.

Figure 7.

The gap junction network of astrocytes and oligodendrocytes. This drawing depicts our model for the GJ channels that connect astrocytes (A) to oligodendrocytes (O) and other astrocytes. A/A junctions contain Cx30/Cx30 and Cx43/Cx43 channels; O/A junctions contain Cx32/Cx30 and Cx47/Cx43 channels. Homotypic Cx32/Cx32 channels connect layers of myelin sheath at the paranode and incisures (data not shown). Cx29 hemichannels are localized to the adaxonal membrane of oligodendrocytes, apposing the axon.