Inversion of both gating polarity and CO₂ sensitivity of voltage gating with D3N mutation of Cx50

Abstract

The effect of CO 2-induced acidification on transjunctional voltage (Vj) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO2 application greatly decreased the Vj sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO2 also affected the kinetics of Vj dependent inactivation of junctional current (Ij), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO2 sensitivity of chemical gating such that even CO2 concentrations as low as 2.5% significantly lowered junctional conductance (Gj). With Cx50 channels Gj dropped by 78% with a drop in intracellular pH (pHi) to 6.83, whereas with Cx50-D3N channels Gj dropped by 95% with a drop in pHi to just 7.19. We have previously hypothesized that the way in which Vj gating reacts to CO2 might be related to connexin's gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO2 on Vj gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes GAP JUNCTIONS are regions of cell contact that mediate the exchange of small cytosolic molecules via cell-cell channels. A gap junction channel results from the extracellular interaction of two hemichannels (connexons), each a hexamer of connexin proteins. Connexins (Cx) contain four transmembrane domains, two extracellular loops, a cytoplasmic loop, a short NH 2 terminus (NT), and a COOH terminus of variable length. The sequences of cytoplasmic loop and COOH terminus vary significantly among the members of the connexin family, whereas those of the other domains are relatively well conserved (see Ref. 14 for review).Gap junction channels are gated by transjunctional voltage (V j ; 27) and increased intracellular [Ca 2ϩ ] (10, 25) or [H ϩ ] i (28, 33) via molecular mechanisms that are still poorly defined (see Ref. 12 for review). At least two V j -sensitive gates have been identified: fast and slow. On the basis of their behavior at the single channel level, fast V j gate and chemical gate are believed to be distinct: the former closes rapidly (Ͻ1 ms) but incompletely, leaving a 20 -30% residual conductance, whereas the latter closes slowly (8 -10 ms) but completely (5). In contrast, slow V j gate and chemical gate are likely to be the same (5,17,20). Slow and fast V j gates are in series, and each hemichannel appears to have both gates. The slow gate closes at the negative side of V j in all connexin channels tested, whereas the polarity of the fast V j gating mechanism varies among connexin channels (see Ref. 8 for review).In the past, chemical and V j gating have been studied almost exclusively by testing chemicals and V j gradients, respectively, whereas minimal attention has been ...

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure

“…For example, replacement of Asp3 with asparagine in Cx50 also resulted in an inversion of the polarity of V j gating [35]. Interestingly, this substitution also affects the sensitivity of Cx50 channels to CO 2 [35]. …”

“…It is interesting that the critical residues implicated in determining these various physiological properties are among the most divergent amino acids within the NT domain (see [26, 32, 35]). Although overall the sequence of this domain is quite conserved, the substitutions confer many of the characteristic properties that differentiate channels formed of different connexins.…”

Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression

“…Intracellular pH of the oocytes was measured as described by Sasaki et al (1992) and Peracchia and Peracchia (2005). Briefly, the fluorescein derivative pH indicator BCECF B-1151 (Molecular Probes, Eugene, OR, USA) was injected into oocytes in amounts sufficient to reach an intraoocyte concentration of 100 lM (Peracchia and Peracchia 2005).…”

“…Briefly, the fluorescein derivative pH indicator BCECF B-1151 (Molecular Probes, Eugene, OR, USA) was injected into oocytes in amounts sufficient to reach an intraoocyte concentration of 100 lM (Peracchia and Peracchia 2005). After the injection, the oocytes were incubated for 40 min and then placed in a built-in chamber, with the vegetative pole facing the fluorescent beam and held in place using a thin stainless steel wire.…”

Functional roles of the amino terminal domain in determining biophysical properties of Cx50 gap junction channels

“…However, the extent and sensitivity of V j -gating of different gap junction channels can vary drastically (Burt and Spray, 1988; Barrio et al, 1991; Verselis et al, 1994; Purnick et al, 2000). Earlier studies on Cx26, Cx32 and other connexins have indicated that among several domains, the NT domain and the residue within the NT domain play a crucial role in gating polarity, the properties of V j -gating and unitary conductance (Rubin et al, 1992; Verselis et al, 1994; Purnick et al, 2000; Musa et al, 2004; Peracchia and Peracchia, 2005; Srinivas et al, 2005; Dong et al, 2006; Gemel et al, 2006; Verselis and Srinivas, 2008; Kronengold et al, 2012). The molecular basis of V j -gating is not fully understood.…”