Functional requirement for a highly conserved charged residue at position 75 in the gap junction protein connexin 32

Abstract

Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg(75) position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel G(j)-V(j) relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels.

FIGURE 1.

Localization of Cx32WT and selected mutants at the Arg⁷⁵ position expressed in HeLa cells. HeLa cells were transiently transfected with DNA coding for the noted variants of Cx32 and stained with mouse anti-connexin antibodies in conjunction with an Alexa Fluor 594 secondary. As shown, Cx32WT and the R75L mutant show elongated puncta at intercellular appositions (arrows), consistent with gap junction plaques. R75P, R75Q, and R75L all exhibit a pattern of staining consistent with localization to the Golgi compartment. Note that the expression level for the R75W mutant is very low. Scale bar, 20 μm.

FIGURE 2.

Plaque formation by Cx32WT and mutants at the Arg⁷⁵ position. Neuro2a cells were transfected with DNA coding for the noted forms of Cx32 and stained with mouse anti-connexin antibodies in conjunction with an Alexa Fluor 594 secondary. As shown, Cx32WT and all studied mutants except R75W show elongated puncta at intercellular appositions (arrows), consistent with gap junction plaques. Note that the expression level for the R75W mutant is very low. Scale bar, 15 μm.

FIGURE 3.

Representative current traces (a, c, and e) and G_j-V_j relations (b, d, and f) for R75P, R75Q, and R75W expressed in Neuro2a cells and paired heterotypically with Cx32 WT. The steady-state G_j-V_j relations are markedly shifted to the left with steady-state conductances at V_j = 0 that are markedly reduced compared with the conductance at −100 mV, consistent with substantially reduced open probabilities at V_j = 0. Steady-state G_j-V_j relations for each of these parings show very small G_min, suggesting that these channels gate to a nonconducting (or very low conductance) state. Because the Cx32 hemichannel is predicted to be fully open at V_j > −40 mV, the low open probabilities at large positive V_j values are likely ascribable to the mutant hemichannel.

FIGURE 4.

Representative current traces (a, c, e, and g) and G_j-V_j relations (b, d, f, and h) for Cx32WT, R75K, R75H, and R75L expressed in Neuro2a cells and paired homotypically. The R75K and R75H mutants show steady-state conductance voltage relations similar to those for WT, whereas the R75L mutant shows a markedly increased G_min.

FIGURE 5.

Representative current traces (a, c, and e) and G_j-V_j relations (b, d, and f) for R75H, R75K, and R75E expressed in Neuro2a cells and paired with Cx32 WT, and G_j-V_j relations (g, h, and i) for R75L, R75A, and R75V expressed in Neuro2a cells and paired heterotypically with Cx32 WT. The R75H and R75K mutants show relatively symmetric steady-state conductance voltage relations. The conductance voltage relation for the R75E, R75L, R75A, and R75V mutants show marked shifts in their G_j-V_j relations, although the shift for R75L appears to be less than those for R75E, R75A, and R75V.