Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

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Abstract
α-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick α7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu172) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby β-sheet formed by the β7, β9, and β10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the β7 and β10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the β7/β9/β10-sheet as a result of introduction of a disulfide bond between the β strands would alter activation by agonists. Using a nondesensitizing α7 mutant background (L247T), we identified one mutant pair, K144C + T198C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca2+, Ba2+, or Sr2+) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the α7 antagonist methyllycaconitine and were abolished when Glu172 was mutated to glutamine. When the K144C + T198C pair was expressed in wild-type α7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into β7/β9/β10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the β-sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.