Biophysical Properties of CX40 Mutants Linked to Atrial Fibrillation

Abstract

Gap junctions provide a direct intercellular pathway for cell-to-cell signalling and impulse conduction. The three Cx40 mutations (A96S, M136V and G38D) are associated with atrial fibrillation and retain the ability to form functional channels. The biophysical properties of mutant gap junctions were determined in transiently transfected HeLa and N2A cells. All three mutants exhibited an array of macroscopic coupling ranging from 0.5 to 30 nS and similar voltage dependences (Boltzmann fit: Vj,o = 49, 48, 52 mV, and gj,min=0.20, 0.22, 0.2 for A96S, M136V and G38D, respectively) comparable to wild-type Cx40. However, unitary conductance of G38D channels was 1.76 fold higher than the wild-type Cx40 channel (∼220 pS versus ∼125 pS). The A96S and M136V mutants exhibited unitary conductances comparable to the wild-type Cx40 (130-140 pS).The channel permeability was achieved using simultaneous measurement of junctional conductance (gj) and intercellular transfer of a fluorescent probe. All three mutants transferred anionic Lucifer Yellow (LY). The G38D channels exhibited ∼9 fold higher anionic LY permeability relative to the ubiquitous cation K+ (LY/K+) when compared to the wild-type Cx40 (0.017 versus 0.002), while A96S LY transfer was similar to wild-type (0.003). In contrast, G38D channels were almost impermeable to cationic EthBr (0.001), suggesting that G38D is responsible for altered channel selectivity. Conversely, A96S and M136V channels exhibited enhanced EthBr permeability (0.037 and 0.047 versus 0.013 for wild-type Cx40). Altered conductive and permeability properties of mutant channels suggest an essential role for biochemical and electrical coupling in cardiac tissues. These properties may contribute to regional variability in conduction velocity, cell excitability (ionic-metabolic misbalance), and therefore may be implicated in mechanisms of reentry arrhythmias.Supported by NIGMS 088181 and 088180.