Atrial fibrillation-associated connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Abstract

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (Vj) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of Vj gating properties and increased the gap junction channel conductance (γj) by 20-30 pS. While co-expression of the two mutations had similar effects on Vj gating, it synergistically increased γj by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.

Keywords: Atrial fibrillation; Channel conductance; Connexin40; Gap junctions; Hemichannels.

Figure 1

G38D and M163V are produced and form gap junction plaques similarly to WTCx40 in HeLa cells. A, Membrane topology of Cx40 with the locations of G38D and M163V indicated by circles. B, Immunoblot detection of WTCx40, G38D, and M163V in homogenates of transiently transfected HeLa cells. C, Immunofluorescent detection of Cx40 in transiently transfected HeLa cells shows that WTCx40, G38D, and M163V localize to appositional membranes (arrows) as well as within the cytoplasm. Bar, 10 μm.

Figure 2

Gap junctions formed by G38D, M163V, and the co-expressed mutants show altered V_j-dependent gating. A, Plots show the G_j-V_j relationships for homomeric WT (black), G38D (red), and M163V (green) gap junctions and co-expressed (heteromeric) G38D+M163V (orange) (The Boltzmann parameters for the fitted curves are listed in Table 1.). B, Confocal micrographs of double-label immunolocalization of G38D-mcherry (red) and M163V-GFP (green) in transiently co-transfected HeLa cells show extensive co-localization which appears yellow in the merged image. Bar, 8 μm.

Figure 3

Single channel conductances differ for WTCx40, G38D, and M163V gap junctions. A, Whole cell currents (I₁ and I₂) recorded simultaneously from WTCx40 N2a cell pair during a +40 mV V_j step applied to cell 1. Current amplitudes are indicated by dashed lines in the I₂ trace (I_j=-ΔI₂). B, I_j-V_j relationship for WTCx40 channels was generated from channel current amplitudes determined by Gaussian fits of the all points current histogram (not shown) for each 30 s V_j pulse. The mean slope conductance (γ_j ) was 149±2 pS. C, Whole cell currents for a G38D channel during a −30 mV V_j pulse. D, I_j-V_j relationship for G38D. The mean slope γ_j was 178±2 pS. E, Whole cell currents for a M163V channel during a +50 mV V_j pulse. F, I_j-V_j relationship for M163V. The mean slope γ_j was 169±2 pS. G, Whole cell currents from a G38D+M163V expressing N2a cell pair illustrating a main channel conductance state of >220 pS and a <70 pS subconductance state. H, The mean channel current-voltage relationship for G38D+M163V gap junction illustrates its main open state slope γ_j of 218±5 pS.

Figure 4

Hemichannel activity of G38D mutant Cx40 channels. A, Single sweep whole cell current recordings from parental N2a, WTCx40, G38D, M163V, and G38D+M163V N2a cells in zero CaCl₂ saline during a V_m staircase protocol from −80 to +80 mV. High conductance channel openings and closings were evident only in cells expressing G38D. B, G38D hemichannel activity recorded during a +20 to −40 mV V_m step illustrates the activation and deactivation of this channel by depolarizing and hyperpolarizing V_m steps, respectively. C, Current-voltage relationship of the G38D hemichannel measured from three V_m step protocol experiments. The slope conductance, γ_hc, was 271±10 pS.