P.12.5 Subunit specific effects of the invariant AChR Cys-loop Aspartate residues on gating and AChR expression

Abstract

The signature Cys-loop at the interface between binding and gating domains of AChR is crucial for coupling agonist binding to gating but the mechanism is not fully understood. An invariant Cys-loop Asp is present at equivalent positions in all subunits of Cys-loop superfamily receptors of all species, but its functional significance is not known. After discovering that a phenotype-determining Asp140Asn in the AChR δ subunit causes a severe congenital myasthenic syndrome (CMS), we introduced the same mutation into the AChR α, β, and e subunits and examined the expression, burst openings, and gating efficiency of each mutant in HEK cells. We employed mutation analysis, mutagenesis, single-channel recordings, and mutant cycle analysis using wild-type and mutant receptors expressed in HEK cells. Compared to wild-type, the respective protein expressions of the α, β, δ and e mutants were 54%, 79%, 21%, and 100%; the corresponding burst durations were 7%, 18%, 55%, and 62%; and the corresponding gating efficiencies were 1%, 23%, 75%, and 108%. To understand why α Asp138Asn has the greatest effects on gating, we examined its thermodynamic interactions with nearby residues by mutant cycle analysis. This revealed that corresponding αAsp138 is functionally linked to nearby αGln208 which is conserved in mammalian α-subunits and is adjacent to the invariant αArg209 which was previously shown to be essential for rapid and efficient gating. (1) The invariant Cys-loop Asp residues contribute to receptor expression, burst duration, and gating efficiency in a subunit-specific manner. (2) The α-subunit Asp has profound effects on burst length and gating efficiency whereas δ-Asp predominantly affects AChR expression. (3) Mutant cycle analysis indicates that αAsp138 is an essential link in the chain of conformational changes that couple agonist binding to gating. (4) The δAsp140Asn causes CMS mainly by reducing AChR expression.