Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease

Abstract

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

Fig. 1

Topography of the connexin 32 subunit and location of mutants described in this communication. See Results for further details.

Fig. 2

Representative current traces and average G_j–V_j relations for 32WT and three mutants. Fig. 2a, c, e, and g. Xenopus oocytes were injected with the noted mRNA's and paired homotypically as described in the Methods. Both cells were voltage clamped at −30 mV and cell 2 was stepped between −120 and +120 mV in 10 mV increments. Junctional currents for 32WT, H94Q, R15Q, and S85C recorded from cell 1 are shown. Only traces in 20 mV increments from ±20 to ±120 are shown for clarity. Current traces were analyzed as described in the Methods to allow for determination of the instantaneous and steady state G_j–V_j relations for each cell pair. Fig. 2b, d, f, and h. Average instantaneous and steady state G_j–V_j relations for 32WT and three mutants. Data such as shown in Fig. 2a, c, e, g, and i were analyzed as described in the Methods, averaged and plotted as mean±S.E.M. The smooth curves approximating the steady state data correspond to the curves generated by fitting the data to a Boltzmann distribution as described in the Methods. Each point in each G_j–V_j plot is the average value as determined in three to eight independent experiments. Filled squares — steady state conductances; hollow triangles — instantaneous conductances.

Fig. 3

Average G_j–V_j relations for four mutants paired heterotypically with 32WT or 26WT. Figs. 3a to c. Xenopus oocytes were injected with the noted mRNA's and paired heterotypically with Cx32WT. Both cells were voltage clamped at −30 mV and the cell expressing the 32WT was stepped between −120 and +120 mV in 10 mV increments. Current traces were analyzed as described in the Methods, averaged and plotted as mean±S.E.M. The smooth curves approximating the steady state data correspond to the curves generated by fitting the data to a Boltzmann distribution. Figs. 3d and e. Representative current traces and average instantaneous and steady state conductance voltage relations for 32WT paired heterotypically with 26WT were determined as noted above in Fig. 2. Figs. 3f to h. Xenopus oocytes were injected with the noted mRNA's and paired heterotypically with 26WT. Junctional currents were obtained and analyzed as noted above. The resulting conductance voltage relations were averaged and plotted as mean±S.E.M. Individual points are connected for clarity. Each point in each G_j–V_j plot is the average value as determined in three to eight independent experiments. Filled squares — steady state conductances; hollow triangles — instantaneous conductances.

Fig. 4

Representative current traces and G_j–V_j relations for G12S paired homotypically and heterotypically with 26WT. Figs. 4a and b. Representative current traces and G_j–V_j relations for G12S paired homotypically. Current traces were analyzed as described in the Methods; the smooth curve approximating the steady state data corresponds to the curve generated by fitting the data to a Boltzmann distribution. Figs. 4c and d. Representative current traces and G_j–V_j relations for G12S paired heterotypically with 26WT. Individual points are connected for clarity. For a and c only traces in 20 mV increments from ±20 to ±120 are shown. Filled squares-steady state conductances; hollow triangles-instantaneous conductances. Current traces and G_j–V_j relations are representative of findings in at least three independent experiments.

Fig. 5

Representative current traces and average G_j–V_j relations for two mutants paired heterotypically with 32WT or 26WT. Figs. 5a and c. Junctional current traces for R15W and H94Y paired heterotypically with Cx32. For clarity, only traces in 20 mV increments from ±20 to ±120 (or ±10 to ±110 for R15W) are shown. Current traces were analyzed as described in the Methods section to allow for determination of the G_j–V_j relations for each cell pair. Figs. 5b and d. Average G_j–V_j relations for R15W and H94Y paired heterotypically with Cx32. Data such as shown in Figs. 5a and c was analyzed as described in the Methods, averaged and plotted as mean±S.E.M. Each point in each G_j–V_j plot is the average value as determined in three to eight independent experiments. The smooth curves approximating the steady state data correspond to the curves generated by the Boltzmann fits. Figs. 5e and g. Junctional current traces for R15W and H94Y paired heterotypically with 26WT. For clarity, only traces in 20 mV increments from ±20 to ±120 are shown. Current traces were analyzed as described in the Methods section to allow for determination of the G_j–V_j relations for each cell pair. Figs. 5f and h. Average G_j–V_j relations for R15W and H94Y paired heterotypically with Cx32. Current traces were analyzed as described in the Methods, averaged and plotted as mean±S.E.M. Individual points are connected for clarity. Each point in each G_j–V_j plot is the average value as determined in three to eight independent experiments.

Fig. 6

Determination of the steady state G_j–V_j relation for the cell–cell channel formed by heterotypic pairing of oocytes expressing V139M with oocytes expressing 32WT or 26WT. Fig. 6a. Both cells were voltage clamped at −30 mV and the cell expressing the 32WT was stepped between +120 and −120 mV in 10 mV increments as described in the Results. Fig. 6b. Current traces such as those shown in 6a were analyzed as described in the Methods, averaged and plotted as mean±S.E.M. The smooth curves approximating the steady state data correspond to the curves generated by fitting the data to a Boltzmann distribution. Because of very slow closure at V_j=0, current traces could not be normalized to a prepulse prior to being analyzed. Fig. 6c Xenopus oocytes were injected with mRNA for either 26WT or V139M and paired heterotypically. Both cells were voltage clamped at 230 mV and the cell expressing 26WT was successively stepped from +120 to −120 mV in 10 mV increments as described in the Results. Fig. 6d. Current traces such as those shown in Fig. 6c were analyzed as described in the Methods, averaged and plotted as mean±S.E.M. Individual points are connected for clarity. Fig. 6e. Junctional current traces recorded in response to the voltage paradigm pictured in the inset. Both cells were initially clamped to −30 mV. Prior to each test pulse the cell expressing 26WT was subjected to a 45 seconds conditioning pulse such that V_j=+100 mV, closing virtually all cell–cell channels, followed by a 45 s interval at V_j=0 to allow the 26WT hemichannels to reopen. 40 seconds activating test pulses from V_j=−120 mV to V_j=+20 mV were applied. Fig. 6f. Conductance voltage relation determined for the data shown in Fig. 6e. Each activating trace was fit to a sum of exponentials as described in the Methods, to determine the steady state current at that voltage. These currents were then divided by the V_j and plotted to yield Fig. 6g. Individual points are connected for clarity. For a, c and e only traces in 20 mV increments from ±20 to ±120 are shown.