Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46

Abstract

Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional coupling induced by Cx46. In contrast, expression of D3Y or L11S failed to inhibit junctional conductance induced by Cx50. To examine effects of the D3Y and L11S mutations on hemichannel activity, hemichannel currents were measured in connexin cRNA-injected oocytes. Oocytes expressing D3Y exhibited reduced hemichannel activity as well as alterations in voltage gating and charge selectivity while oocytes expressing L11S showed no hemichannel activity. Moreover, coexpression of mutant with wild-type Cx50 or Cx46 gave rise to hemichannels with distinct electrophysiological properties, suggesting that the mutant connexins were forming heteromeric channels with wild-type connexins. These data suggest D3Y and L11S cause cataracts by similar but not identical mechanisms.

Keywords: cataract; connexin; gap junction; intercellular communication.

Fig. 1.

Wild-type (WT) and mutant connexin 46 (Cx46) form gap junction plaques. Immunolocalization with Cx46 antibodies shows that wild-type Cx46 (A), Cx46D3Y (B), and Cx46L11S (C) all form gap junctions in transfected HeLa cells along the plasma membrane between expressing cells. White arrows indicate gap junctions immunostained red, which are shown enlarged in the bottom panel. Calibration bar, 10 μm.

Fig. 2.

D3Y and L11S fail to induce gap junctional coupling. A–C: bar graphs showing the junctional conductances (means ± SE) in pairs of Xenopus oocytes expressing Cx46, D3Y, and L11S or heterotypic combinations of wild-type and mutant lens connexins as determined by double whole cell voltage clamp. The total amount of cRNA injected into each oocyte was held constant. All oocytes were injected with antisense oligonucleotides (AS) to block endogenous Cx38 junctional currents. The number of pairs is indicated in parentheses. *P < 0.001 compared with Cx46-injected oocyte pairs; **P < 0.001 compared with heterotypic Cx50/Cx46 pairs.

Fig. 3.

Heterotypic Cx46/D3Y and Cx50/D3Y gap junctional channels show asymmetrical transjunctional voltage (V_j) gating properties. A–C: representative families of junctional current traces recorded from oocyte pairs expressing Cx46/Cx50 (A), Cx46/D3Y (B), or Cx50/D3Y (C). D–F: plots of normalized steady-state junctional conductance (G_jss) vs. transjunctional voltage for Cx46/Cx50 (D; n = 4), Cx46/D3Y (E; n = 6), and Cx50/D3Y (F; n = 6). Positive and negative V_j refers to depolarization and hyperpolarization, respectively, of the cell indicated on the right side of the pairing designation. Points represent means ± SE.

Fig. 4.

D3Y and L11S act as strong dominant negative inhibitors of wild-type Cx46 but not wild-type Cx50 gap junctional channels. Bar graphs show the junctional conductances (means ± SE) in pairs of Xenopus oocytes expressing Cx46, Cx50, or a 1:1 mixture of wild-type and mutant connexins as determined by double whole cell voltage clamp. The amount of Cx46 or Cx50 injected into each oocyte was held constant. The number of pairs is indicated in parentheses. *P < 0.001 compared with Cx46-injected cell pairs; **P < 0.001 compared with AS-injected cell pairs.

Fig. 5.

Cells expressing D3Y show alterations in hemichannel gating. A–C: representative examples of hemichannel currents in single oocytes injected with D3Y (A) or Cx46 (B) cRNA or no RNA (AS, C). The bath solution contained choline chloride Ringer with zero added calcium. The hemichannel currents were recorded in response to a series of voltage-clamp steps from +80 mV to −100 mV in decrements of 20 mV followed by a short pulse to −80 mV. Holding potential = −10 mV. Data were corrected for leakage. Dashed line represents zero current. D: steady-state current-voltage relationships for oocytes injected with cRNA for D3Y (○), Cx46 (■), or AS alone (▲) using the paradigm described in A. E: conductance-voltage relationships for D3Y (○, n = 6) and Cx46 (■, n = 6). Initial amplitudes of tail currents were measured at −80 mV after 20-s pulses to different potentials, normalized to the maximum amplitude of the tail current, and plotted as a function of prepulse potential.

Fig. 6.

Summary of the reversal potentials (V_rev) of Cx46 (A) and D3Y (B) in external solutions containing different KCl concentrations. Symbols represent means of measured reversal potentials ± SE (n = 3–8). The solid lines were calculated according to the Goldman-Hodgkin-Katz (GHK) equation. The dashed lines show the predictions for ideally cationic and anionic conductances. The intracellular permeant ion activity was assumed to be 93 mM for K⁺, 9.4 mM for Na⁺, and 35 mM for Cl⁻ (12). V_rev was determined using a ramp protocol as described in methods.

Fig. 7.

Oocytes coinjected with D3Y and Cx46 induce large hemichannel currents with novel voltage gating properties. A and C: representative examples of hemichannel currents in single oocytes injected with D3Y + Cx46 (A) or L11S + Cx46 (C) cRNA. The bath solution contained choline chloride Ringer with zero added calcium. The hemichannel currents were recorded in response to a series of voltage-clamp steps from +80 mV to −100 mV in decrements of 20 mV followed by a short pulse to −80 mV. Holding potential = −10 mV. Data were corrected for leakage. Dashed line represents zero current. B and D: conductance-voltage relationships for D3Y + Cx46 (n = 4; B) and L11S + Cx46 (n = 4; D). Initial amplitudes of tail currents were measured at −80 mV after 20-s pulses to different potentials, normalized to the maximum amplitude of the tail current, and plotted as a function of prepulse potential. For comparison, the conductance-voltage curve for Cx46 (dashed line) is shown.

Fig. 8.

Heteromeric (D3Y + Cx50) and (L11S + Cx50) hemichannels demonstrate altered gating properties. A–C: representative examples of hemichannel currents in single oocytes injected with Cx50 (A) or L11S + Cx50 (B) or D3Y + Cx50 (C) cRNA. The bath solution contained choline chloride Ringer with zero added calcium. The hemichannel currents were recorded in response to a series of voltage-clamp steps from +80 mV to −100 mV in decrements of 20 mV followed by a short pulse to −80 mV. Holding potential = −10 mV. Data were corrected for leakage. Dashed line represents zero current. D: conductance-voltage relationships for Cx50 (■ squares, n = 7), Cx50 + Cx46 (○, n = 6), D3Y + Cx50 (△, n = 5), L11S + Cx50 (▼, n = 4). Initial amplitudes of tail currents were measured at −80 mV after 20-s pulses to different potentials, normalized to the maximum amplitude of the tail current, and then plotted as a function of prepulse potential.

Fig. 9.

A and B: effect of substituting tyrosine for Asp-3 in the homology-modeled structure of Cx46. Asp-3 forms a ring of negatively charged side chains at the narrowest part of the hemichannel in wild-type Cx46 (A). In the D3Y mutant, the narrowest part of the channel is occluded by the larger tyrosine rings (B). Residues 1–10 and 12–14 are shown as light gray space filling structures; residue 3 is shown as dark gray space filling structures, with oxygen in red and nitrogen in blue. All hydrogens are omitted for clarity. C: effect of substituting serine for Leu-11 in the homology-modeled structure of Cx46. Leu-11 sits at the center of a hydrophobic cluster, surrounded by Phe-6, Leu-7, Leu-10, Leu-93, and Leu-97. Mutation of Leu-11 to a serine would disrupt this cluster, and may easily lead to large-scale reorganization of this region. Since this cluster forms a portion of the interface between two long helices within each monomer, and between helices on adjacent monomers, the serine mutation could also have long-range effects on the conformation of the hemichannel.