Received for publication February 8, 2006.
Revised March 13, 2006.
Accepted for publication March 13, 2006.

Role of the Outer Sheet in Divalent Cation Modulation of 7 Nicotinic Receptors

Abstract

Alpha-7 nicotinic acetylcholine receptors (AChR) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick 7 AChR this modulation involves a conserved glutamate in loop 9 (E¹⁷²; Galzi et al., 1996) that undergoes agonist-dependent movements during activation (Lyford et al., 2003). From these observations we hypothesized that movements of the nearby sheet formed by the 7, 9, and 10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis we examined functional properties of cysteine mutations of the 7 and 10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the 7/9/10 sheet due to introduction of a disulfide bond between the strands would alter activation by agonists. Using a non-desensitizing 7 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 ACh. Divalent-evoked currents were blocked by the 7 antagonist methyllycaconitine (MLA) and were abolished when E¹⁷² was mutated to glutamine. When the K¹⁴⁴C + T¹⁹⁸C pair was expressed in wild-type 7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into 7/9/10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite co-agonists.

Key words: Nicotinic cholinergic, Ion channel regulation, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches

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