Roles of nicotinic acetylcholine receptor beta subunits in function of human alpha4-containing nicotinic receptors

Abstract

Naturally expressed nicotinic acetylcholine receptors (nAChR) containing alpha4 subunits (alpha4*-nAChR) in combination with beta2 subunits (alpha4beta2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. beta4 subunits are also richly expressed and colocalize with alpha4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, alpha4beta4-nAChR also may exist and play important functional roles. In this study, properties were determined of human alpha4beta2- and alpha4beta4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human alpha4beta4-nAChR have approximately 4-fold higher amplitude than those mediated via human alpha4beta2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at alpha4beta4-nAChR than at alpha4beta2-nAChR. Cytisine and lobeline serve as full agonists at alpha4beta4-nAChR but are only partial agonists at alpha4beta2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional alpha4beta2- and alpha4beta4-nAChR. Whole-cell current responses show stronger inward rectification for alpha4beta2-nAChR than for alpha4beta4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR beta2 or beta4 subunits can combine with alpha4 subunits to generate two forms of alpha4*-nAChR with distinctive physiological and pharmacological features. Diversity in alpha4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.

Figure 1. Morphology and transgene expression in SH-EP1 cells

A, phase-contrast photomicrograph of transfected SH-EP1 cells during patch-clamp recording. B, RT-PCR analysis of α4, β2 or β4 nAChR subunit transcripts or GAPDH internal controls (C) from wild-type (WT) SH-EP1 cells or from cells cotransfected with α4 plus either β2 (α4β2) or β4 (α4β4) subunits. Lanes labelled ‘RT(−)’ are for samples from SH-EP1-hα4β4 cells processed after omission of the RT step. Sizes of RT-PCR products resolved on a 1% agarose gel are calibrated using a 100-bp DNA ladder (New England BioLabs Inc., Beverly, MA, USA). C, immunolabelling of nAChR α4 subunits on SH-EP1-hα4β2 cells. Ca, low-magnification, phase-contrast view of SH-EP1-hα4β2 cells. Cb, high-magnification, fluorescence view of a SH-EP1-hα4β2 cell labelled with rat monoclonal antibody 299 against the nAChR α4 subunit. Cc, non-transfected SH-EP1 cells labelled with the same antibody as in B. Cd, a SH-EP1-hα4β2 cell labelled with secondary antibody only. Note the bright, punctate, specific labelling (arrows in panel Cb) on the surface of SH-EP1-hα4β2 cells and the absence of specific labelling on non-transfected cells and SH-EP1-hα4β2 cells exposed only to secondary antibody. Bars in Ca and Cb represent 5 μm; bar in Cb applies to panels Cb–Cd.

Figure 2. Electrophysiological properties of α4β2- and α4β4-nAChR

Low (3 or 1 μm, A) or high (100 μm, B) concentrations of nicotine were applied to induce whole-cell inward currents in transfected SH-EP1 cells stably expressing human α4β2- (Aa and Ba) or α4β4- (Ab and Bb) nAChR. Superimposed traces from each row are shown in the right column. C, bar graphs summarizing results of replicate studies (31 cells each) illustrating differences between α4β2- (filled bars) and α4β4- (open bars) nAChR responses in net current density (a), the ratio of steady-state to peak components (b), the rising time to peak whole-cell current (c) and whole-cell current decay constant (d). Data were averaged from 31 cells tested for Fig. 2 Ca-c, and 18 cells tested for α4β4-nAChR and 20 cells tested for α4β2-nAChR in Fig. 2Cd, and vertical bars represent s.e.m. **P < 0.01 for difference between α4*-nAChR subtypes. All data presented in Fig. 2C were obtained from whole-cell currents induced by equipotent concentrations of 3 μm nicotine (α4β2-nAChR) or 1 μm nicotine (α4β4-nAChR).

Figure 3. Functional rundown of α4β2- and α4β4-nAChR

Using K⁺ electrodes at a V_H of −60 mV, 3 or 1 μm nicotine was repetitively applied for 4 s at 1-min intervals to SH-EP1 cells expressing either α4β2- (A) or α4β4- (B) nAChR, respectively. Peak current components from whole-cell current traces are plotted against trace number in (C) for α4β2- (▪) or α4β4- (•) nAChR. Each symbol represents the average from six cells tested, and vertical bars represent s.e.m. *P < 0.05; **P < 0.01.

Figure 4. Nicotine concentration–response relationships for α4β2- and α4β4-nAChR

Five superimposed whole-cell current traces elicited in response to nicotine exposure (0.01–100 μm) are shown for cells expressing either α4β2- (A) or α4β4- (B) nAChR. C, nicotine alone (*) concentration–response curves plotted for absolute peak current values for α4β2- (▪) or α4β4- (•) nAChR show differences in current amplitudes. D, nicotine concentration–response curves plotted for peak currents normalized to those evoked in response to 100 μm nicotine alone (*) show differences at α4β2- (▪) or α4β4- (•) nAChR in agonist potency. In C and D, each symbol represents the average from 6–8 cells, and vertical bars represent s.e.m.

Figure 5. Concentration–response curves for cytisine, lobeline and epibatidine acting at α4β2- and α4β4-nAChR

Peak current responses of α4β2- (left column) or α4β4- (middle column) nAChR evoked by cytisine (A; ♦ or ⋄), lobeline (B; ▴ or ▵), or epibatidine (EPBD) (C; • or ○) are plotted after being normalized to the response evoked by 100 μm nicotine (▪ or □; indicated by *) or (right column) responses of both α4*-nAChR subtypes to those drugs normalized to the maximal response to a given drug are plotted. Each symbol represents the average from 5 to 6 cells tested, and vertical bars represent standard errors.

Figure 6. Antagonism of α4β2- and α4β4-nAChR function

3 μm (A) or 1 μm (B) nicotine (∼EC₅₀ concentration)-induced whole-cell currents obtained from exposure to SH-EP1-hα4β2 (left) or -hα4β4 (right) cells alone or in the presence of different concentrations of DHβE are superimposed. C and D, effects of coexposure to DHβE (♦, ⋄), mecamylamine (MEC; ▪, □), hexamethonium (HEXA; •, ○), d-tubocurarine (d-TC; ▴, ▵) or methyllycaconitine (MLA; ▾, ▿) on nicotine-evoked whole-cell peak current responses of α4β2- (3 μm nicotine, C) or α4β4-nAChR (1 μm nicotine, D) are plotted as concentration–response curves. Each symbol represents the average from 5–6 cells, and vertical bars represent s.e.m.

Figure 7. Comparison of antagonist action at α4β2- and α4β4-nAChR

Nicotine concentration–response curves alone or in the presence of 0.3 μm DHβE (Aa, α4β2-nAChR; Ab, α4β4-nAChR), 1 μm MEC (Ba, α4β2-nAChR; Bb, α4β4-nAChR) or 0.5 μm HEXA (Ca, α4β2-nAChR; Cb, α4β4-nAChR) are superimposed. All nicotinic responses were normalized to the current induced by 100 μm nicotine alone (*). Each symbol represents the average from 5–8 cells, and vertical bars represent s.e.m.

Figure 8. Current-voltage (I–V) relationships for α4β2- and α4β4-nAChR

A, whole-cell peak currents evoked by 100 μm nicotine (indicated by black horizontal bar) at different V_H values (−80, 0, +60 mV) are shown above current–voltage response curves normalized to the response to nicotine at −100 mV (*) for α4β2- (Aa; □ in Ac) or α4β4- (Ab; ▪ in Ac) nAChR. Each symbol represents the average from six cells, and vertical bars (in c) represent s.e.m. B, sample traces for I–V curve analyses for α4β2- (a) and α4β4- (b) nAChR whole-cell current responses assessed using a higher (30 mm) concentration of Na⁺ in the pipette solution evoked by 1 mm ACh (indicated by black horizontal bar) at different V_H values (−80, 0, +80 mV) are shown above current–voltage response curves normalized to the response to nicotine at −80 mV (*) for α4β2- (Ba; ○ in (Bc) or α4β4- (Bb; • in Bc) nAChR.