Regulation of neuronal connexin-36 channels by pH

Abstract

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH(i) since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

Fig. 1.

Alkalinization reduces the electrical coupling mediated by neuronal Cx36 channels. Cell pairs coupled through rat connexin43 (rCx43) and human connexin36 (hCx36) junctions that were exposed first to control medium (pH_o 7.40) and then superfused with acidic or basic solutions (bars) before again switching them to the control medium while the junctional conductance (G_j; black) was monitored and the pH_i (pH_i; red) was measured with SNARF-1. G_j was normalized relative to its value at the resting pH_o. (A) Acidification typically causes a rapid and reversible uncoupling of Cx43 (Upper) but it slightly increases the Cx36 G_j between the oocyte (Middle; n = 12, P < 0.05) and N2A cell pairs (Bottom; n = 10, P < 0.05). (B) Alkalinization produces a mild increase in Cx43 coupling (upper; n = 9, P < 0.05) yet it reversibly reduces the G_j for Cx36. Note that the G_j in the Cx36 oocytes tends to decrease somewhat before it increases upon acidosis (arrow), while a small increase in G_j typically precedes the uncoupling with alkalosis (arrowhead). Data in Figs. 1–5 represent the mean values ± SE. (n = 9–12).

Fig. 2.

pH_i sensitivity of human Cx36 junctions. The curve was constructed with the steady state values of G_j in oocytes (●) and in N2A cells (○) after stabilization of the pH_i value following each pH stimulus (n = 12). The average G_j/pH_i relationship was fitted with a four-parameter sigmoidal function of the form: G_j = G_jmax/(1+exp(slope(pHi-pK_H)), where G_j is maximal at the acidic extreme (G_jmax = 1.17) and decreases in the alkalotic direction with a midpoint channel activity parameter pK_H of 7.86.

Fig. 3.

Alkalinization-induced uncoupling is common among Cx36 orthologues. Oocytes were injected with the RNAs encoding for mouse Cx36 (mCx36), chicken Cx35.1 (chCx35.1), perch Cx35 (pCx35), and skate Cx35 (sCx35). (A) Intracellular acidification induced small increases in the G_j of mCx36 and pCx35 and a moderate rise in chCx35.1, while the G_j of sCx35 decreased slightly (n = 9, P < 0,05). (B) By contrast, alkalization caused a marked reduction in the G_j of mCx36 and pCx35 and a moderate decrease in chCx35.1 and sCx35. Arrows and arrowheads as in Fig. 1.

Fig. 4.

Mechanisms of pH gating. Human Cx36 was mutated in its three cytoplasmic domains (Left Column) by creating the H18Q substitution in the NT-domain (NT-H18Q), the deletion of residues S101 to Q155 in the cytoplasmic loop (CL-delS101-Q155), and a truncation of the CT-domain at position 282 (CT-L282stop). The NT-H18Q and CT-L282stop mutations abolished the uncoupling at alkalosis (A) and the mild increase in G_j following acidosis of wild type junctions (HCx36wt) (B). Moreover, these mutations induced a novel pH regulation characterized by minor increases and decreases with alkalosis and acidosis, respectively (n = 12, P < 0.05). The CL-delS101-Q155 mutation reduced the G_j for alkalosis to a similar level as in the wild type and induced a large increment in the G_j following acidosis.

Fig. 5.

Comparison of the pH_i sensitivity of mutant and wild-type hCx36 channels. The pH_i/G_j relationships of the data shown in Fig. 4 were constructed by plotting the changes in G_j along the time course of alkalinization and subsequent re-acidification, and vice versa. (A) The dynamic pH_i/G_j relationships of wild type junctions (solid black line) did not differ significantly from the steady state curve shown in Fig. 2 (dotted line). The pH_i sensitivity of NT-H18Q (green) and CT-L282stop (red) channels is similar, with minor albeit saturating changes in G_j in the opposite pH direction to those of the wild type. The curves can be described with a regular Hill equation (solid lines) of the form G_j = G_jmax/(1+pK_a/pH_i)^h + G_jmin and with the parameters for NT-H18Q of maximal G_jmax and minimal G_jmin conductance of 1.16 and 0.7, a midpoint channel activity pK_a = 7.15, and with a Hill coefficient h = 3.7 and the parameters of G_jmax = 1.14 and G_jmin = 0.8, a pK_a = 6.94 and a h = 3.3 for the CT-L282stop mutant. The theoretical titration curve for the alkalotic mechanism suppressed by these two mutants corresponds to a Hill equation with a G_jmax = 1.36, a G_jmin near zero, a pK_a = 7.57 and a h = 2.6 (broken black line). (B) The curve for CL-delS101-Q155 channels (blue) shows a large increase of G_j for acidosis in concordance with the displacement of pK_H 7.86 to 6.68 (Inset).