Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease

Abstract

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Figure 1

Representative current traces for oocytes injected with either 0.1 μl of a 175 ng/μl solution of mRNA for Cx32(S85C) (AntiCx38) (A), mRNA for Cx32WT (B), or antiCx38 alone (C). Currents evoked by depolarizing the Cx32(S85C)-injected cells were larger and activated at smaller voltages than did those from either the Cx32WT- or antiCx38-injected cells. Traces in 20 mV increments from +10 mV to +90 mV for Cx32(S85C) and +30 mV to +90 mV for Cx32WT and antiCx38 are shown.

Figure 2

Current–voltage relations for oocytes injected with antiCx38 or with 0.1 μl of a 175 ng/μl solution of mRNA for Cx32(S85C) or Cx32WT. The points for Cx32WT (*) and Cx32(S85C) (▿) are the currents measured at the end of 20-s pulses from +10 to +80. Traces for antiCx38 (■) are in response to 20-s pulses from −70 mV to voltages ranging from +20 to +80 mV. Activating currents are seen in Cx32(S85C) cells at voltages above +10 mV, whereas both Cx32WT- and AntiCx38-injected cells begin to show outward currents at higher voltages. Although no significant differences between antiCx38- and Cx32WT-injected cells were detected by using the Kruskal–Wallis test with Dunn's post test, the Cx32WT-injected cells did show a trend toward slightly larger outward currents. Each curve is representative of data from at least 19 oocytes (means ± SEM).

Figure 3

Properties of Cx32(S85C) hemichannels. (A) Effect of acidification on voltage-activated currents in oocytes injected with mRNA for Cx32(S85C). An oocyte bathed in MND96 with Ca²⁺ was pulsed between −70 mV and +30 mV. After the third pulse (at the second arrow), the bath was perfused with CO₂-saturated MND96 with Ca²⁺, which caused a rapid and complete loss of voltage-activated current. The conductance recovered when the bath was perfused with control MND96 with Ca²⁺ (third arrow). (B) Effect of reduced-bath Ca²⁺ on voltage-activated currents in oocytes injected with mRNA for Cx32(S85C). A single oocyte bathed in MND96 with 1.8 mM Ca²⁺ was pulsed between −70 mV and +30 mV. After the third pulse (at the second arrow), the bath was perfused with MND96 with 200 μM Ca²⁺, with a resulting 2 1/2-fold increase in magnitude of voltage-evoked current.

Figure 4

Expression of Cx32(S85C) in oocytes is lower than that of Cx32WT. (A) Western blot analysis of total protein extracts from 13 pooled oocytes injected with mRNA for Cx32(S85C), mRNA for Cx32WT, or antiCx38 alone. The blot was probed with antibody to Cx32, as described in Methods. As shown, the levels of Cx32 protein in the Cx32(S85C)-injected oocytes are between 1/2 and 1/4 that of the Cx32WT-injected oocytes. (B) Western blot analysis of surface-membrane protein extracts from the same oocytes shown in A [mRNA for Cx32(S85C), mRNA for Cx32WT, or antiCx38 alone]. Membrane proteins were isolated by reacting intact oocytes with the membrane impermeant reagent NHS-S-S-Biotin and recovered with neutravidin-coated beads (see Methods). The amount of protein in the membrane was substantially greater for Cx32WT than for Cx32(S85C).