The unique α4+/-α4 agonist binding site in (α4)3(β2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (α4)2(β2)3 subtype

Abstract

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Fig. 1.

Schematic diagrams of concatenated HS and LS α4β2-nAChR isoform pentameric constructs (HSP and LSP, respectively). (A) At the cDNA level, individual subunits are joined using linkers that encode AGS repeats. Each linker also contains a unique restriction site, noted at the base of (A). These sites allow rapid removal and replacement of individual subunits by a simple restriction digestion and ligation approach (Carbone et al., 2009). For both HSP and LSP, long-recognized agonist binding pockets form at the interfaces between the principal (+) faces of α4 subunits in positions 2 and 4, and the complementary (−) faces of β2 subunits in positions 1 and 3, respectively. The functional pharmacology implications of the further, LSP-unique, agonist binding pocket between the (+) face of α4 position 5 and the (−) face of α4 position 4 are explored in this study; site-directed mutant α4 subunits were substituted in positions 4 and 5 of the original LSP construct. (B) Schematic of an assembled HSP α4β2-nAChR, showing the arrangement of the subunit + and – interfaces, the resulting pair of agonist binding pockets, and the linkers. (C) Schematic of an assembled LSP α4β2-nAChR. Note that much of the structure is similar to the HSP isoform, but that an α4 subunit is substituted for the β2 subunit in position 5. This gives rise to the additional agonist binding site shown between the fourth and fifth subunits of LSP α4β2-nAChR.

Fig. 2.

Ligand-specific activation or desensitization of α4β2-nAChR. SH-EP1-hα4β2 cells stably expressing functional, human α4β2-nAChRs from loose subunits were subjected to acute challenges for 5 minutes with the indicated drugs at the specified concentrations (abscissa; log M scale) for determination of specific ⁸⁶Rb⁺ efflux (defined in Materials and Methods) (ordinate; % of control response to a full agonist, 1 mM carbamylcholine). (A) Acute response to Saz-A (●). (B) Acute response to A-85380 (●). (C) Acute response to Saz-A after 10-minute pretreatment with 3.16 nM Saz-A (♦) or A-85380 (◇). (D) Acute response to A-85380 after 10-minute pretreatment with 3.16 nM Saz-A (♦) or A-85380 (◇). Results were fit to unconstrained, one- or two-site logistic equations. The derived logEC₅₀ values, Hill coefficients, and fractional contributions of each site (in the case of two-site fits) are reported in the text.

Fig. 3.

Acute Saz-A and A-85380 stimulation of HSP versus LSP α4β2-nAChR expressed in X. laevis oocytes. X. laevis oocytes expressing human HSP and LSP α4β2-nAChR constructs were acutely stimulated with the indicated range of Saz-A (A) or A-85380 (B) concentrations. Values are the mean ± S.E.M. (n = 3 individual determinations), and are reported as the percentage of maximally efficacious ACh control responses (experimental details in Materials and Methods). Results were fit to unconstrained one-site logistic equations. The derived agonist logEC₅₀, Hill coefficient, and efficacy values are reported in the text.

Fig. 4.

Saz-A pretreatment effects on HSP versus LSP α4β2-nAChR function. X. laevis oocytes injected with mRNA encoding either wild-type HSP α4β2-nAChR or wild-type LSP α4β2-nAChR were tested for the effects of Saz-A (5-minute) pretreatment on subsequent, acute, agonist stimulation. Initial control stimulation was performed for 1 second, using a maximally effective concentration of ACh (30 µM for HSP, 1 mM for LSP). Oocytes were then exposed to Saz-A (3.16 nM) for 5 minutes, and the ACh challenge was repeated (1-second stimulation, coapplied with 3.16 nM Saz-A); typical traces are shown. Saz-A pretreatment greatly reduced HSP responses (peak response diminished to 17.6 ± 2.1%; mean ± S.E.M., n = 4). In contrast, Saz-A pretreatment diminished LSP peak responses to 33.3 ± 3.0% (mean ± S.E.M., n = 4). The difference in response to Saz-A pretreatment between HSP and LSP was significant (unpaired two-tailed t test, t = 4.21 with 6 degrees of freedom; P < 0.01).

Fig. 5.

ACh activation of wild-type versus mutant LSP α4β2-nAChR. X. laevis oocytes injected with unmodified or mutant LSP mRNA were exposed to acute ACh challenges (1 second, concentrations specified on the x axis; log M scale). Filled symbols show concentration-response relationships determined for each LSP variant when exposed to acute ACh stimulation. Note that the proportion of HS-phase function is enhanced for every LSP mutant compared with the unmodified LSP construct. Open symbols depict concentration-response determinations for the same constructs, when exposed to a range of ACh concentrations after 5-minute preincubation with Saz-A (3.16 nM, 5 minutes). Pretreatment with Saz-A reduced HS-phase function of all LSP variants to such an extent that it could no longer be fit as part of a two-site model, leaving only LS-phase function. Log EC₅₀ values for HS- and LS-phase responses, fractions of HS- versus LS-phase function, and statistical analyses are reported in Table 1. Points are the mean ± S.E.M. (n = 6–12).

Fig. 6.

Pretreatment with Saz-A eliminates the HS-phase of ACh-induced LSP function, and changes the EC₅₀ of LS-phase responses. X. laevis oocytes injected with unmodified HSP (squares) and LSP (circles) mRNA were exposed to acute ACh challenges (1 second, concentrations specified on the x axis; log M scale), before (filled symbols) and after (open symbols) pretreatment with Saz-A (3.16 nM, 5 minutes). All concentration-response data are normalized to the maximum response measured for each concentration series, in each oocyte (mean ± S.E.M., n = 8–12). For HSP α4β2-nAChRs, no change in ACh EC₅₀ value was seen after preincubation with Saz-A. For LSP α4β2-nAChRs, the HS-phase of the CRC (below approximately 10^−5.5 M) was abolished by preincubation with Saz-A, whereas the potency of the LS-phase response was reduced. Log EC₅₀ values for HS- and LS-phase responses, fractions of HS- versus LS-phase function, and statistical analyses are reported in Table 1.

Fig. 7.

Effects of changing Saz-A preincubation concentration on LSP and HSP ACh-induced responses. X. laevis oocytes injected with unmodified LSP (A) and HSP (B) mRNA were first exposed to a maximally effective ACh control challenge, followed by pretreatment with Saz-A (5 minutes, concentrations indicated in legends). Concentration-response data were then collected using a series of acute ACh challenges (1 second, concentrations specified on the x axis; log M scale, see Materials and Methods for details). All concentration-response data were normalized to the initial control stimulation, in each oocyte (mean ± S.E.M., n = 3). For LSP α4β2-nAChR, Saz-A preincubation reduced (1 nM) and then abolished (> 3.16 nM) HS-phase responses. ACh EC₅₀ values and magnitudes of LS-phase responses were unaffected by increasing Saz-A preincubation concentrations (3.16–31.6 nM; pharmacological parameters reported in the text). For HSP α4β2-nAChRs, ACh responses were reduced by 1 nM Saz-A preincubation but, in contrast to LSP responses, no function remained after 31.6 nM Saz-A preincubation.

Fig. 8.

LSP α4(+)/(−)α4 interface mutation effects on maximum peak ACh-induced function (I_max) and nAChR cell-surface expression. X. laevis oocytes were injected with mRNAs encoding either unmodified or mutant LSP constructs (loop A, Y126A; loop B, W182A and Y184A; loop C, Y223A and Y230A; loop D, W88A), or an unmodified HSP construct (see Materials and Methods for details). (A) Maximum peak ACh-induced function (I_max) was determined for each construct. I_max is significantly reduced by several of the mutations compared with the unmodified LSP construct, and the HSP construct also has a significantly lower I_max value than that recorded for the wild-type LSP construct. *P < 0.05; ***P < 0.001. (B) nAChR protein expression at the surface of X. laevis oocytes was determined using an [¹²⁵I]mAb295 binding assay. To note, binding of two [¹²⁵I]mAb295 molecules per LSP construct and three per HSP construct (reflecting the different β2 subunit numbers in each nAChR isoform; see Materials and Methods) was assumed. Although some apparent variation in nAChR cell-surface expression was observed across the tested constructs, this did not reach the level of statistical significance. (C) I_max values were normalized to the amounts of nAChR cell-surface expression for each construct. By applying the HS-phase function fractions calculated for each construct (Table 1), it was possible to determine the absolute amount of HS- and LS-phase function for each construct. Significant changes are noted as follows: **P < 0.01; ***P < 0.001 for HS-phase function; and ⁺P < 0.05; ⁺⁺P < 0.01 for overall function (I_max). To note, the HSP result was not included in the overall function comparison for (C), since this construct produces only HS-phase function. Details of the analysis applied, and parameter values determined, are supplied in Table 2.

Fig. 9.

Comparison of Saz-A pretreatment effects on LS-phase function across LSP variants. X. laevis oocytes were injected with unmodified and mutant LSP mRNAs. ACh concentration-response experiments were performed to determine maximal LS-phase function before and after pretreatment with Saz-A (3.16 nM, 5 minutes). The percentage of post-treatment versus pretreatment LS-phase function is displayed for each LSP variant construct used in this study. Values are the mean ± S.E.M. of 6–8 individual determinations collected from three separate experiments. One-way ANOVA showed no significant differences across the LSP variants (F_6,14 = 1.85; P = 0.16).