Modulation of Cx46 hemichannels by nitric oxide

Abstract

Gap-junction hemichannels are composed of six protein subunits (connexins). Undocked hemichannels contribute to physiological autocrine/paracrine cell signaling, including release of signaling molecules, cell-volume regulation, and glucose uptake. In addition, hemichannels may be pathologically activated by dephosphorylation and cell-membrane depolarization. Such hemichannel opening may induce and/or accelerate cell death. It has been suggested that connexin43 (Cx43) hemichannels are sensitive to redox potential changes and that one or more intracellular cysteines is/are important for this process. Cx46 is expressed in the lens, and its dysfunction induces cataract formation. It contains six cysteines in the extracellular loops, one in the fourth transmembrane helix, and two in the COOH-terminal domain. The latter may be susceptible to oxidation by nitric oxide (NO), which could be involved in cataract formation through cysteine S-nitrosylation. Here we report studies of the effects of the NO donor S-nitrosoglutathione (GSNO) on the electrical properties and fluorescent-dye permeability of wild-type Cx46 and mutant hemichannels expressed in Xenopus laevis oocytes. GSNO enhanced hemichannel voltage sensitivity, increased tail-current amplitude, and changed activation and closing kinetics in Cx46 and Cx46-CT43 (Cx46 mutant in which the COOH terminus was replaced with that of Cx43), but not in Cx46-C3A (Cx46 in which the intracellular and transmembrane helix 4 cysteines were mutated to alanine). We conclude that Cx46 hemichannels are sensitive to NO and that the NO effects are mediated by modification of one or more intracellular cysteines. However, it is unlikely that NO induces cataract formation due to the hemichannel activation, because at normal resting potential, NO had no major effects on Cx46 hemichannel permeability.

Fig. 1.

Schematic representation of connexin46 (Cx46) and Cx46 mutants. The diagram (extracellular-surface up) shows the location of Cys residues, denoted by circles. Cx46, wild-type rat Cx46; Cx46-C3A, Cx46 with intracellular and M4 Cys mutated to Ala; Cx46ΔCT, Cx46 with truncation of the COOH-terminal domain at position 239; Cx46-CT43, chimera in which the Cx43 COOH terminus (thicker line) was fused to Cx46ΔCT. In addition to Cys218, it has 3 additional cysteines at positions 256, 267, and 294.

Fig. 2.

Nitric oxide (NO) alters the electrophysiological properties of Cx46 hemichannels. A–D: typical whole cell current records from Xenopus oocytes expressing wild-type Cx46 and Cx46 mutants. Hemichannel current recordings were obtained under control conditions (ND96 solution plus Ca²⁺ and Mg²⁺) and after exposure to 1 mM S-nitrosoglutathione (GSNO) for 40 min (after NO). Oocytes were clamped to −60 mV, and square pulses from −60 mV to +60 mV (in 10-mV steps) were then applied for 15 s. At the end of each pulse, the membrane potential was returned to −60 mV for 10 s. Arrows point to +60 mV records (see text).

Fig. 3.

Intracellular Cys are necessary for the nitric oxide effect. A–D: current-voltage (I-V) plots of normalized currents recorded in oocytes expressing wild-type Cx46 and Cx46 mutants under control conditions (•) or after exposure to 1 mM GSNO (○). Currents were measured at the end of the 15-s pulses and were normalized to the +60-mV value. The Boltzmann equation (see text) was fitted to the data. Continuous line, control; segmented line, GSNO. See Fig. 2 for additional details.

Fig. 4.

GSNO alters Cx46 and Cx46-CT43 hemichannel tail currents. A–D: typical tail-current records from Xenopus oocytes expressing wild-type Cx46 and Cx46 mutants, before and after exposure to GSNO (after NO). See Fig. 2 for details. E: effect of NO on peak tail-current amplitudes elicited upon transition from +60 mV to −60 mV. Bars are means ± SE of 11 (Cx46), 10 (Cx46-C3A), 4 (Cx46ΔCT), and 9 (Cx46-CT43) experiments. **P < 0.01 and ***P < 0.001. The dotted line denotes control values. F: effects of NO on tail-current kinetics. The data were fit to a two-exponential equation, and the resulting time constants, fast (τ_fast) and slow (τ_slow), are shown as means ± SE of the NO values relative to control. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control.

Fig. 5.

The effect of GSNO on Cx46 and Cx46-CT43 hemichannels is reversed by DTT. Shown are representative whole cell recordings of Cx46 and Cx46-CT43 hemichannel currents after 40-min exposure to 1 mM GSNO in the absence of DTT (GSNO) or after 15-min exposure to 10 mM DTT (GSNO + DTT). See Fig. 2 for details.

Fig. 6.

DTT alters Cx46-CT43 but not Cx46 hemichannel currents. A: representative whole cell recordings of Cx46 and Cx46-CT43 hemichannel currents under control conditions and after a 15-min exposure to 10 mM DTT. B: I-V plots of the normalized currents shown in A. ▵, Cx46; ▴, Cx46-CT43. WT, wild type. The fit of the data after DTT to the Boltzmann equation is depicted by the solid (Cx46) and segmented (Cx46-CT43) lines. See Fig. 2 for details.

Fig. 7.

GSNO affects dye uptake through Cx46 but not Cx46-C3A hemichannels. Oocytes expressing wild-type Cx46 or Cx46-C3A were exposed to 2 mM 5(6)-carboxyfluorescein (CF), 1 mM Lucifer yellow (LY), or 1 mM ethidium bromide (EthBr), indicated at the top, for 40 min, in the absence or presence of 1 mM GSNO. After treatment, the oocytes were washed and sonicated and the dye content was measured by fluorometry. Data depicted are the normalized effects of 1 mM GSNO on dye uptake by oocytes in ND96 (1.8 mM Ca²⁺, open bars) or in the nominal absence of divalent cations (hatched bars). For the normalization, the dye uptake in GSNO was divided by that in the absence of GSNO at normal or low [Ca²⁺]. Data are from 3 independent experiments for each dye (8 oocytes per experiment). *P < 0.05, control condition vs. 1 mM GSNO.