Structural domains involved in the regulation of connexin 36 gap junction channels by general anaesthetics

Abstract

Investigation of mechanisms of general anaesthetics action emerges a great challenge in the design of novel drugs acting on the central nervous system (CNS). Connexin 36 (Cx36) gap junction (GJ) channels are expressed in the CNS and may be involved in general anaesthesia. In order to examine binding of general anaesthetics to Cx36 GJs, protein-ligand docking, coarse-grained molecular dynamics, site-directed mutagenesis and patch-clamp recording of GJ conductance (gj) were used. Protein–ligand docking showed a similar accumulation pattern of hexanol and isoflurane which involved the unique intrasubunit cavity for both molecules only, situated inside of each Cx36 wt subunit in the vicinity of N terminus and transmembrane domains TM1 and TM2 (with residing C87 and C92). The second intrasubunit cavity, predicted in the proximity of the unique one, contained mainly hexanol and isoflurane molecules as well as low quantity of nonanol. The highest accumulation of nonanol was predicted in the intersubunit cavity involving TM2 domain of one Cx36 wt subunit and TM1 and TM4 domains of the neighboring subunit. Cysteines of TM2 and TM4 domains were predicted to be possible elements of the intra- and inter- subunit cavities which could contribute to general anesthetics binding. Patch-clamp recordings of HeLa cells transfected with Cx36 wt and cysteine mutants confirmed the ligand docking results: hexanol and isoflurane stimulated Cx36 wt GJ coupling (gj), while nonanol blocked it. C92V (TM2) weakened the gj stimulatory effect of hexanol and isoflurane while C264S (TM4) reversed it to inhibition. The blocking effect of nonanol was reduced only in C92V (TM2) and C168A (intracellular loop) mutants. Finally, coarse-grained molecular dynamics showed that C264S mutation, in contrast to Cx36 wt, caused hexanol to be adsorbed in the vicinity of major parts of amino acids of N terminus, TM1 and TM2 domains which formed intra- and inter- subunit cavities. Our results implicate pot