Second, mutations at the 4(+)/()4 site of LS isoform 42-nAChR preferentially reduced LS phase function

Second, mutations at the 4(+)/()4 site of LS isoform 42-nAChR preferentially reduced LS phase function. counterparts or vice versa (2HQT and 4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed inXenopusoocytes. Acetylcholine concentration-response curves and maximum function were assessed using two-electrode voltage clamp electrophysiology. Surface expression was measured with125I-mAb 295 binding and was used to determine function/nAChR. If the 4(+)/()2 sites contribute equally to function, making identical 2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, 4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent 4(+)/()2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect. Keywords: electrophysiology, monoclonal antibody, nicotinic acetylcholine receptors (nAChR), receptor structure-function, site-directed mutagenesis, ligand-gated ion channels == Introduction == Nicotinic acetylcholine receptors (nAChR)2are ligand-gated ion channel neurotransmitter receptors. In mammals, they are expressed as pentameric combinations of homologous subunits, translated from 16 different genes (17, 9, 10, 14,,, and ). Functional diversity of nAChR is determined by subunit composition, producing nAChR subtypes with overlapping pharmacological and biophysical characteristics (1). 42*-nAChR are the most prevalent central nervous system (CNS) nAChR subtype, comprising 70% of all rodent CNS nAChR (2) and are implicated in a wide range of normal and pathological functions, including learning, memory, mood, and nicotine dependence among others (317). 42*-nAChR functionally interact with nicotine at concentrations found in smokers and are the target of varenicline, currently the most successful smoking cessation pharmacotherapy (12, 13). Initial studies suggested an (4)2(2)3subunit stoichiometry for these nAChR (18, 19). However , more Aliskiren D6 Hydrochloride recent work indicates that both native and heterologously expressed 42-nAChR can exist in two Aliskiren D6 Hydrochloride isoforms with (4)2(2)3and (4)3(2)2subunit stoichiometries, respectively, displaying high and (predominantly) low sensitivities (HS and LS) to activation by acetylcholine (ACh) (2025). The expression of 42-nAChR isoforms appears to be physiologically significant. For example , multiple epilepsy-associated 4 and 2 subunit mutants alter ratios of HS to LS 42-nAChR isoforms (17, 26), and agonists capable of preferentially stimulating LS 42-nAChR produce distinctive physiological effects (2729). Accordingly, a better understanding of the respective roles of HS and LS 42*-nAChR isoforms is likely to have considerable translational implications. Agonist binding to nAChR is primarily driven by interactions with a set of six peptide loops located at subunit interfaces. Three (loops AC) are contributed from the subunit on the principal or (+)-side of the interface, with the remaining three (loops DF) being contributed from the subunit on the complementary or ()-side of the interface. The ()-side subunit is oriented counterclockwise from the (+)-side subunit when viewed from extracellular space (Fig. 1A) (20). A high degree of conservation of critical agonist binding residues is seen across subunits. This includes residues within interfaces that do not harbor conventionally recognized canonical Aliskiren D6 Hydrochloride (+)/()-type agonist binding pockets (3034). Both 42-nAChR isoforms host a pair of canonical orthosteric, high affinity 4(+)/()2 agonist binding interfaces, but the LS (4)3(2)2-nAChR isoform also contains a unique, CPB2 non-canonical, 4(+)/()4 agonist-binding site (3436). This 4(+)/()4 site has lower affinity for ACh or nicotine than the 4(+)/()2 site, making it responsible for the intrinsically biphasic ACh concentration-response profile of the LS (4)3(2)2-nAChR isoform. This complex CRC distinguishes it from the HS (4)2(2)3-nAChR isoform, which lacks an 4(+)/()4 subunit interface and produces monophasic CRCs (3436). == FIGURE 1 . == Schematic illustration of E-loop chimera construction using concatenated LS (4)3(2)2and HS (4)2(2)3isoforms of nAChR. A, top, diagram representing the linked LSP and HSP 42-nAChR isoforms. Illustrated are subunit positions and agonist binding pockets (filled circlesandtriangle) formed from a principal (+) and E-loop-containing complementary ()-face as observed from extracellular space. This convention is used in subsequent figure captions to indicate the positions of.