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¹ Division of Neurology
² Division of Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013-4496, USA
³ Division of Science, Chandler-Gilbert Community College, Chandler, AZ 85225, USA
⁴ Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724-5017, USA

	Abstract

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Naturally expressed nicotinic acetylcholine receptors (nAChR) containing 4 subunits (4*-nAChR) in combination with ß2 subunits (4ß2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. ß4 subunits are also richly expressed and colocalize with 4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, 4ß4-nAChR also may exist and play important functional roles. In this study, properties were determined of human 4ß2- and 4ß4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human 4ß4-nAChR have 4-fold higher amplitude than those mediated via human 4ß2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at 4ß4-nAChR than at 4ß2-nAChR. Cytisine and lobeline serve as full agonists at 4ß4-nAChR but are only partial agonists at 4ß2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional 4ß2- and 4ß4-nAChR. Whole-cell current responses show stronger inward rectification for 4ß2-nAChR than for 4ß4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR ß2 or ß4 subunits can combine with 4 subunits to generate two forms of 4*-nAChR with distinctive physiological and pharmacological features. Diversity in 4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.

(Received 2 June 2006; accepted after revision 5 July 2006; first published online 6 July 2006)
Corresponding author J. Wu: Neurophysiology Laboratory, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013-4496, USA. Email: jie.wu{at}chw.edu

	Introduction

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Nicotinic acetylcholine receptors (nAChR) in mammals exist as a diverse family of molecules composed of different combinations of subunits derived from at least 16 genes (see reviews by Lukas et al. 1999; Jensen et al. 2005). nAChR are prototypical members of the ligand-gated ion channel superfamily of neurotransmitter receptors and models used to establish concepts pertaining to mechanisms of drug action, synaptic transmission, and structure and function of transmembrane signalling molecules. The most abundant form of heteromeric nAChR in the brain contains 4 and ß2 subunits (4ß2-nAChR; Whiting & Lindstrom, 1987; Flores et al. 1992; Gopalakrishnan et al. 1996; Lindstrom, 1996) although additional subunits may integrate into these complexes. 4ß2-nAChR bind nicotine with high affinity, and respond to levels of nicotine found in the plasma of smokers (Benowitz et al. 1989; Hsu et al. 1995; Lindstrom, 1996; Fenster et al. 1997; Lukas et al. 1999; Jensen et al. 2005; Lukas, 2006). They have been implicated in nicotine self-administration, reward, and dependence, and in diseases such as Alzheimer's disease and epilepsy (Picciotto et al. 1995; Cordero-Erausquin et al. 2000; Jensen et al. 2005).

nAChR ß4 subunit mRNA colocalizes in different species with 4 subunit mRNA in numerous brain regions (Winzer-Serhan & Leslie, 1997; Quik et al. 2000) implicated in complex behaviours, including nicotine dependence. Knock-out studies suggest that nAChR containing ß2 or ß4 subunits in mouse brain play different roles. For example, in nAChR ß2(–/–) animals, nicotine fails to elicit striatal dopamine release, fails to increase (at concentrations similar to those found in the arterial blood of human smokers) discharge frequency of midbrain dopaminergic neurons, and fails to elicit self-administration, suggesting roles for nAChR-containing ß2 subunits in nicotine reinforcement (Picciotto et al. 1998). Although ß2 subunit substitution in ß4(–/–) animals can prevent the perinatally lethal effects of autonomic failure seen in ß2(–/–)/ß4(–/–) mice, ß4(–/–) mice still exhibit autonomic dysfunction (Wang et al. 2003), have heightened resistance to nicotine-induced seizures (Kedmi et al. 2004), and have decreased nicotine withdrawal symptoms (Salas et al. 2004). Heterologous expression in Xenopus oocytes of rat nAChR 2, 3 or 4 subunits in pairwise combination with either ß2 or ß4 subunits produces nAChR having differing nicotinic agonist binding affinities attributable to the ß subunit assembly partner (Parker et al. 1998). As part of a literature demonstrating functional heterologous expression of functional nAChR-containing ß4 subunits, rat nAChR ß2 or ß4 subunits influence kinetics of whole-cell current decay phase (Fenster et al. 1997) and sensitivity to competitive nicotinic antagonists and Zn²⁺ modulation (Harvey & Luetje, 1996). Moreover, heterologously expressed, rat 2ß4- or 3ß4-nAChR show an unusual functional potentiation in the presence of D-tubocurarine at lower concentrations (1–10 µM) than those needed to inhibit nicotinic responses (Cachelin & Rust, 1994).

To establish a foundation for studies of the possible roles of human 4ß4-nAChR in addition to 4ß2-nAChR in mediation of classic cholinergic excitatory neurotransmission, in modulation of neurotransmitter release, and/or in neuropsychiatric diseases, and to enhance understanding of roles played by ß subunits in nAChR structure and function, patch-clamp recording of whole-cell currents was used to undertake a systematic comparison of functional properties of human 4ß2- and 4ß4-nAChR heterologously expressed in the SH-EP1 human epithelial cell line. Our results show clear distinctions between these nAChR in desensitization kinetics, affinities and efficacies of nicotinic ligands, and current–voltage relationships, demonstrating important influences of ß subunits on 4*-nAChR function.

	Methods

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Expression of human neuronal 4ß2- and 4ß4-nAChR in SH-EP1 human epithelial cells

Human 4 and ß2 or ß4 subunits (kindly provided by Dr Ortrud Steinlein or Jon Lindstrom) were subcloned into pcDNA3.1-zeocin and pcDNA3.1-hygromycin vectors, respectively, and transfected using established techniques (Puchacz et al. 1994; Peng et al. 1999, 2005) into native nAChR-null SH-EP1 cells (Lukas et al. 1993) following the approach taken to create the SH-EP1-h4ß2 cell line (Eaton et al. 2003) in order to generate the SH-EP1-h4ß4 cell-line (Eaton et al. 2000). Briefly, three million SH-EP1 cells in 0.5 ml of 20 mM Hepes, 87 mM NaCl, 5 mM KCl, 0.7 mM NaH₂PO₄, 6 mM dextrose, pH 7.05, in an electroporation cuvette, were mixed with 100 µg of vector DNA containing the ß4 subunit insert. Samples were subjected to electroporation (Bio-Rad Gene Pulsar model 1652076, Richmond, CA, USA) at 960 µF and 200 V. After electroporation, cells were suspended to 5 ml in complete medium (Bencherif & Lukas, 1993), and 1 ml aliquots were added to 12 ml aliquots of medium in each of five 100 mm dishes. Forty-eight hours later, positive selection was initiated in medium supplemented with 0.4 mg ml^–1 hygromycin (Calbiochem, La Jolla, CA, USA). The polyclonal pool of surviving cells was harvested and subjected to a second round of transfection via electroporation with vector DNA containing the 4 subunit insert and selected for zeocin (0.5 mg ml^–1; Invitrogen, Carlsbad, CA, USA) resistance beginning 48 h later. Colonies of surviving cells were picked, subcloned by limiting dilution, and expanded before being screened for radioligand binding and functional evidence for 4ß4-nAChR expression, which led to selection of the clone designated as the SH-EP1-h4ß4 cell line. Cells were maintained as low passage number (1–26 from our frozen stocks) cultures, to ensure stable expression of phenotype, maintained in medium augmented with 0.5 mg ml^–1 zeocin and 0.4 mg ml^–1 hygromycin, and passaged once weekly by splitting just-confluent cultures 1/10 to maintain cells in proliferative growth.

RNA preparation and reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated from the cells growing at approximately 80% confluence in a 100 mm culture dish using 2 ml of Trizol reagent (Bethesda Research Laboratories, Gaithersburg, MD, USA). Prior to RT-PCR, RNA preparations were treated with amplification-grade, RNase-free DNase (Bethesda Research Laboratories, Gaithersburg, MD, USA) in order to remove residual genomic DNA contamination. Typically, 1 µg of RNA was incubated with 1 unit of DNaseI in a 10 µl reaction at room temperature for 15 min, and then the DNase was inactivated by addition of 1 µl of 25 mM EDTA and incubated at 65°C for 10 min. RT was carried out using 0.8 µg of DNA-free total RNA, oligo d(T)_{12 – 18} primer, and the Superscript II Preamplification system (Bethesda Research Laboratories, Gaithersburg, MD, USA) in a 20 µl reaction. At the end of the RT reaction, reverse transcriptase was deactivated by incubation at 75°C for 10 min, and RNAs were removed by adding 1 unit of RNaseH followed by incubation at 37°C for 30 min. A typical PCR was performed using 2 µl of cDNA preparation, 1 µl of 10 µM each of 5'- and 3'-gene-specific primers, 1 µl of 10 mM dNTP, and 2.5 units of RedTaq (Sigma, St. Louis, MO, USA) in a 50 µl reaction. The primers used in the amplification stage were designed and synthesized based on published gene sequences (GenBank accession number: 4 NM000744, ß2 NM009602, ß4 NM000750; GAPDH BC026907). The primer sequences and their predicted product sizes are: 4 sense 5'-GAATGTCACCTCCATCCGCATC-3', 4 antisense 5'-CCGGCA(A/G)TTGTC(C/T)TTGACCAC-3' (product size 790 bp); ß2 sense 5'-CGGCTCCCTTCCAAACACA-3', ß2 antisense 5'-GCAATGATGGCGTGGCTGCTGCA-3' (product size 754 bp); ß4 sense 5'-TCTGGTTGCCTGACATCGTG-3', ß4 antisense 5'-GGGTTCACAAAGTACATGGA-3' (product size 847 bp); GAPDH sense 5'-CGTATTGGGCGCCTGGTCACCAG-3', GAPDH antisense 5'-GTCCTTGCCCACAGCCTTGGCAGC-3' (product size 624 bp). Amplification reactions were carried out in a RoboCycler (Stratagene, La Jolla, CA, USA) for 35 amplification cycles at 95°C for 1 min, 55°C for 90 s, and 72°C for 90 s, followed by an additional 4-min extension at 72°C. One-tenth of each RT-PCR product was then resolved on a 1% agarose gel, and sizes of products were determined based on migration relative to mass markers loaded adjacently.

Immunolabelling of nAChR 4 subunit in SH-EP1 cells

nAChR subunits on the surface of SH-EP1- h4ß2 and non-transfected SH-EP1 controls were immunolabelled with a rat monoclonal antibody against the nAChR 4 subunit (clone 299; Covance, Berkeley, CA, USA). Live, unfixed, non-permeabilized cells were exposed (4°C, 1 h) to primary antibody diluted in PBS containing 5% bovine serum albumin (BSA; Sigma Chemical, St Louis, MO, USA), followed by fluorescent, antirat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Cells were then fixed (2% formaldehyde in PBS, 4°C, 30 min), dehydrated (methanol, –30°C, 5 min), air-dried, and mounted in ProLong antifade medium (Molecular Probes/Invitrogen, Carlsbad, CA, USA).

Samples were examined with a Zeiss Axiovert microscope using a 100x, high-NA objective for fluorescence, and digital images were collected with a Photometrics cooled CCD camera. Images were processed with Photoshop (Adobe Systems, San Jose, CA, USA) using the same settings for different samples.

Patch-clamp recordings

Conventional patch-clamp whole-cell current recordings coupled with techniques for fast application and removal of drugs (U-tube) were applied in this study as previously described (Wu et al. 1996, 2002, 2004a and b,; Zhao et al. 2003). Briefly, cells plated on polylysine-coated 35-mm culture dishes were placed on the stage of an inverted microscope (Olympus IX7, Lake Success, NY, USA) and continuously superfused with standard external solution (2 ml min^–1). Glass microelectrodes (1.5 x 100 mm, Narishige, East Meadow, NY, USA) were made in two steps using a vertical electrode puller (P-830, Narishige, East Meadow, NY, USA). Electrodes with a resistance of 3–5 M between the pipette and external solutions were used to form tight seals (>1 G) on the cell surface, until suction was applied in order to convert to conventional whole-cell recording. Thereafter, the recorded cell was lifted and then voltage clamped at a holding potential (V_H) of –60 mV (unless specifically mentioned), and ionic currents in response to application of nicotinic ligands were measured (Axopatch 200B amplifier, Axon Instruments, Union City, CA, USA). Whole-cell access resistance was less than 20 M before series resistance compensation. Both pipette and whole-cell current capacitances were minimized, and series resistance was routinely compensated to 80%. Typically, data were acquired at 10 kHz, filtered at 2 kHz, displayed and digitized online (Digidata 1322 series A/D board, Axon Instruments), and stored to hard drive. Data acquisition and analyses of whole-cell currents were done using Clampex9.2 (Axon Instruments), and results were plotted using Origin 5.0 (OriginLab Corp., North Hampton, MA, USA) or Prism 3.0 (GraphPad Software, Inc., San Diego, CA, USA). Concentration–response curves were fitted to the Hill equation. nAChR acute desensitization was analysed for decay time constant (), peak current (I_p), and steady-state current (I_s) using fits to the expression

(1)

or to its bi-exponential variant as appropriate, using data from 90% to 10% of the period between the peak amplitude of the inward current and the termination of the typical 4 s period of agonist exposure. Replicate determinations of (a measure of the rate of acute desensitization) and I_s/I_p (a measure of the extent of acute desensitization) are presented as means ± standard errors (S.E.M.), and comparisons of and I_s/I_p values across different conditions were analysed for statistical significance using the Student's t test (paired or independent). All experiments were performed at room temperature (22 ± 1°C).

Solutions and drug application

The standard external solution contained (mM): 120 NaCl, 3 KCl, 2 MgCl₂, 2 CaCl₂, 25 D-glucose, 10 Hepes, pH adjusted to 7.4 with Tris-base. In some experiments using ACh as an agonist, 1 µM atropine sulphate was added to the standard solution to exclude any possible influences of muscarinic receptors, but the results were the same as those obtained in the absence of atropine (data not shown). Three types of pipette solutions were used for conventional whole-cell recording. Electrodes containing (mM): 140 KCl, 4 MgCl₂, 1 CaCl₂, 0.5 EGTA, 4 Na-ATP, 10 Hepes, pH adjusted to 7.2 with KOH (K⁺ electrodes) were used to examine current–voltage relationships. Electrodes containing (mM): 120 CsCl, 30 NaCl, 4 MgCl₂, 1 CaCl₂, 0.5 EGTA, 4 Na-ATP, 10 Hepes, pH adjusted to 7.2 with KOH (Cs⁺ electrodes) were used in some cases to further compare inward rectification between 4ß2- and 4ß4-nAChR. This pipette solution was used for two reasons: (a) replacing K⁺ with Cs⁺ increased recording stability at more positive V_H values, and (b) increasing the Na⁺ concentration (from 4 to 34 mM) showed more clear outward currents at positive V_H values. Electrodes containing (mM): 110 Tris phosphate dibasic, 28 Tris base, 11 EGTA, 2 MgCl₂, 0.1 CaCl₂, 4 Na-ATP, pH 7.3 (Tris⁺ electrodes; Huguenard & Prince, 1992) were used for other experiments, since this pipette solution was reported to prevent receptor functional rundown (Zhao et al. 2003).

To initiate whole-cell current responses, under constant superfusion of the recording chamber, nicotinic drugs were rapidly delivered to the recorded cell using a computer-controlled ‘U-tube’ application system. Using this device, the rising time (10–90%) for junction potential effects induced by applied diluted external solution (50% distilled water) was 8.3 ms, and rising times for 1 mM ACh-induced whole-cell currents (V_H = –60 mV, lifted whole-cell recording) were 10.9 ± 1.1 ms (n = 15) and 39.2 ± 2.1 ms (n = 8) for 4ß2-nAChR and 4ß4-nAChR, respectively. In some experiments, a fast-step drug application system (SF-77B, Warner Ins. Co., Hamden, USA) was used. With minimal movement of it (100 µm), the rising time for junction potential effects was 3.0 ms, and the rising times for 1 mM ACh-induced whole-cell currents (V_H = –60 mV, lifted whole-cell recording) were 10.8 ± 1.2 ms (n = 9) and 33.9 ± 3.2 ms (n = 9) for 4ß2-nAChR and 4ß4-nAChR, respectively. Intervals between drug applications (3 min) were adjusted specifically to ensure stability of nAChR responsiveness (absence of functional rundown), and the selection of pipette solutions used in most of the studies described here was made with the same objective. Drugs used in the present study were (–)nicotine, ACh, epibatidine, cytisine, lobeline, dihydro-ß-erythroidine (DHßE), mecamylamine (MEC), hexamethonium (HEXA), D-tubocurarine (dTC), and methyllycaconitine (MLA) (Sigma Chemical Co., St. Louis, MO, USA).

	Results

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Evidence for expression of nAChR subunits in transfected SH-EP1 cells

SH-EP1 cells exhibit a range of morphologies before (not shown) or after (Fig. 1A) transfection with nAChR subunits. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed expression of human nAChR 4 and ß2 subunit messages in SH-EP1-h4ß2 cells, and expression of human 4 and ß4 subunit messages in SH-EP1-h4ß4 cells (Fig. 1B). By contrast, there was no such expression in the absence of the reverse transcription step or in the untransfected cell host, despite successful amplification of GAPDH message in all of the cells (Fig. 1B). Immunocytochemical staining using a monoclonal anti-nAChR 4 subunit antibody showed bright, punctate, specific labelling on the surfaces of transfected SH-EP1-h4ß2 cells (Fig. 1Cb; see phase contrast image at lower power in Fig. 1Ca) but no specific labelling on untransfected cells (Fig. 1Cc) or SH-EP1-h4ß2 cells exposed only to secondary antibody (Fig. 1Cd). These results indicated appropriate expression of nAChR subunits from cDNAs as messages and as cell surface protein in stably transfected SH-EP1 cells.

View larger version (78K): [in this window] [in a new window]   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 4, ß2 or ß4 nAChR subunit transcripts or GAPDH internal controls (C) from wild-type (WT) SH-EP1 cells or from cells cotransfected with 4 plus either ß2 (4ß2) or ß4 (4ß4) subunits. Lanes labelled ‘RT(–)’ are for samples from SH-EP1-h4ß4 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 4 subunits on SH-EP1-h4ß2 cells. Ca, low-magnification, phase-contrast view of SH-EP1-h4ß2 cells. Cb, high-magnification, fluorescence view of a SH-EP1-h4ß2 cell labelled with rat monoclonal antibody 299 against the nAChR 4 subunit. Cc, non-transfected SH-EP1 cells labelled with the same antibody as in B. Cd, a SH-EP1-h4ß2 cell labelled with secondary antibody only. Note the bright, punctate, specific labelling (arrows in panel Cb) on the surface of SH-EP1-h4ß2 cells and the absence of specific labelling on non-transfected cells and SH-EP1-h4ß2 cells exposed only to secondary antibody. Bars in Ca and Cb represent 5 µm; bar in Cb applies to panels Cb–Cd.

Distinctive properties of functional 4ß2- and 4ß4-nAChR

Whole-cell current recording using Tris⁺ electrodes revealed the expression of functional 4ß2- or 4ß4-nAChR in transfected SH-EP1-h4ß2 or SH-EP1-h4ß4 cells responding to nicotine (Fig. 2). One obvious difference in whole-cell response profiles depending on the nAChR ß subunit assembly partner was the kinetics and extent of whole-cell current decay from peak to steady-state levels during a single exposure to nicotine. We operationally define this process as nAChR acute desensitization. The rate of acute desensitization was much slower for 4ß4-nAChR than for 4ß2-nAChR, and the extent of whole-cell current decline was smaller for 4ß4-nAChR than for 4ß2-nAChR (Fig. 2A and B). Another obvious difference was the higher amplitude of 4ß4-nAChR responses (peak and steady-state) to low (around EC₅₀; see below) or high (100 µM) concentrations of nicotine (Fig. 2A and B). Results obtained from studies of SH-EP1 cells each expressing either 4ß2- (n = 31) or 4ß4-nAChR (n = 31) confirm the higher amplitude responses mediated by 4ß4-nAChR after normalizing inward current to cell capacitance (16.3 ± 1.6 pA pF^–1 current density for 4ß2-nAChR; 41.3 ± 5.7 pA pF^–1 for 4ß4-nAChR; Fig. 2Ca). Across those cells, in the presence of EC₅₀ concentrations of nicotine, the average ratio of steady-state/peak currents was 34 ± 1% for 4ß2-nAChR and 85 ± 2% for 4ß4-nAChR (Fig. 2Cb), and the average time required for an e-fold rise inward current was 59 ± 3 ms for 4ß2-nAChR and 107 ± 6 ms for 4ß4-nAChR, reflecting faster opening of 4ß2-nAChR channels (Fig. 2Cc). The average time constants for e-fold decline in nicotine-induced whole-cell current from peak values were 440 ± 50 ms for 4ß2-nAChR (nicotine 3 µM, n = 20) and 2480 ± 160 ms for 4ß4-nAChR (nicotine 1 µM, n = 18, Fig. 2Cd), reflecting the faster and more extensive acute desensitization of 4ß2- compared to 4ß4-nAChR. These effects were not artifacts of the rate of drug delivery, and the lower amplitude of the peak current response mediated by 4ß2-nAChR was not a consequence of the faster desensitization of those receptors. Whole-cell current densities induced in the presence of a lower concentration of nicotine (100 nM) that produced no clear desensitization of either receptor were 1.99 ± 0.36 pA pF^–1 (n = 7) for 4ß2-nAChR and 4.72 ± 0.66 pA pF^–1 (n = 7, P < 0.01) for 4ß4-nAChR, preserving the >2-fold higher response of 4ß4-nAChR compared to 4ß2-nAChR.

View larger version (23K): [in this window] [in a new window]   Figure 2.  Electrophysiological properties of 4ß2- and 4ß4-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 4ß2- (Aa and Ba) or 4ß4- (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 4ß2- (filled bars) and 4ß4- (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 4ß4-nAChR and 20 cells tested for 4ß2-nAChR in Fig. 2Cd, and vertical bars represent S.E.M. **P < 0.01 for difference between 4*-nAChR subtypes. All data presented in Fig. 2C were obtained from whole-cell currents induced by equipotent concentrations of 3 µM nicotine (4ß2-nAChR) or 1 µM nicotine (4ß4-nAChR).

View larger version (17K): [in this window] [in a new window]   Figure 3.  Functional rundown of 4ß2- and 4ß4-nAChR Using K+ electrodes at a VH of –60 mV, 3 or 1 µM nicotine was repetitively applied for 4 s at 1-min intervals to SH-EP1 cells expressing either 4ß2- (A) or 4ß4- (B) nAChR, respectively. Peak current components from whole-cell current traces are plotted against trace number in (C) for 4ß2- () or 4ß4- (•) nAChR. Each symbol represents the average from six cells tested, and vertical bars represent S.E.M. *P < 0.05; **P < 0.01.

Nicotinic agonist actions at 4ß2- and 4ß4-nAChR

4ß2- and 4ß4-nAChR-mediated whole-cell currents induced by different concentrations of nicotine were recorded using Tris⁺ electrodes at a V_H of –60 mV (Fig. 4A and B). Absolute (Fig. 4C) or normalized (to the response to 100 µM nicotine; Fig. 4D) peak whole-cell current responses to nicotine when plotted as a function of nicotine concentration were sigmoidal and well-fitted by a single-site model of the logistic equation (with no improvement in fit using a two-site model). Fits to the data yielded EC₅₀ values and Hill coefficients of 3.1 µM (2.0–4.7 µM; 95% confidence interval) and 0.78 ± 0.09 (S.E.M.), respectively, for 4ß2-nAChR (n = 10 cells), and 1.3 µM (0.72–2.20 µM) and 1.18 ± 0.27, respectively, for 4ß4-nAChR (n = 7 cells; Fig. 4D). Moreover, peak current amplitude was about 5-fold higher for 4ß4- than for 4ß2-nAChR (Fig. 4C). These findings demonstrated both higher efficacy (Fig. 4C) and affinity (Fig. 4D) for nicotine acting at human 4ß4-nAChR compared to 4ß2-nAChR (Table 1). Whole-cell peak current response profiles for other nicotinic agonists, including cytisine (Fig. 5A), lobeline (Fig. 5B) and epibatidine (EPBD) (Fig. 5C), were obtained and normalized to the maximal response to nicotine (at 100 µM; Fig. 5A–C, columns a and b). These profiles indicate partial (20%) efficacy of cytisine and lobeline (compared to maximal effects of nicotine or ACh) at 4ß2-nAChR, but nearly full or full efficacy at 4ß4-nAChR, whereas epibatidine acts as a full agonist at both 4ß2- and 4ß4-nAChR. When responses to individual agonists were normalized to their own maximal effect at a given 4*-nAChR subtype (Fig. 5A–C, column c), it was evident that EC₅₀ values for cytisine were about 6-fold lower and EC₅₀ values for nicotine, ACh, lobeline or epibatidine were about 2.4- to 3.4-fold lower for actions at 4ß4-nAChR compared to actions at 4ß2-nAChR (Table 1). Rank-order agonist potency (EC₅₀ values and 95% confidence intervals provided in parentheses) is epibatidine (0.100 µM; 0.03–0.36) >> cytisine (1.3 µM; 0.79–2.20) nicotine (3.1 µM; 2.0–4.7) = lobeline (3.1 µM; 2.5–3.9) ACh (4.1 µM; 3.2–5.0) for 4ß2-nAChR, and epibatidine (0.029 µM; 0.020–0.043) > cytisine (0.21 µM; 0.14–0.33) > nicotine (1.3 µM; 0.72–2.20) = lobeline (1.3 µM; 0.73–2.30) ACh (1.7 µM; 1.2–2.4) for 4ß4-nAChR. These results indicate that cytisine or lobeline act on 4ß2-nAChR as partial agonists, but act on 4ß4-nAChR as full agonists, providing a means for distinguishing between 4*-nAChR subtypes in addition to generally higher agonist potency at 4ß4-nAChR (Table 1).

View larger version (22K): [in this window] [in a new window]   Figure 4.  Nicotine concentration–response relationships for 4ß2- and 4ß4-nAChR Five superimposed whole-cell current traces elicited in response to nicotine exposure (0.01–100 µM) are shown for cells expressing either 4ß2- (A) or 4ß4- (B) nAChR. C, nicotine alone (*) concentration–response curves plotted for absolute peak current values for 4ß2- () or 4ß4- (•) 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 4ß2- () or 4ß4- (•) nAChR in agonist potency. In C and D, each symbol represents the average from 6–8 cells, and vertical bars represent S.E.M.

View this table: [in this window] [in a new window]   Table 1.  Profiles for agonist interactions with 4ß2- and 4ß4-nAChR

View larger version (39K): [in this window] [in a new window]   Figure 5.  Concentration–response curves for cytisine, lobeline and epibatidine acting at 4ß2- and 4ß4-nAChR Peak current responses of 4ß2- (left column) or 4ß4- (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 4*-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.

Nicotinic antagonist actions at 4ß2- and 4ß4-nAChR

The effects of selected nicotinic antagonists on function of 4ß2- and 4ß4-nAChR elicited by EC₅₀ concentrations of nicotine were assessed using Tris⁺ electrode recording. DHßE coapplied with nicotine reduced both peak and steady-state current responses of both 4ß2- and 4ß4-nAChR in a concentration-dependent manner (Fig. 6A and B). Similar studies evaluated concentration-dependent effects of other antagonists on function of 4ß2- and 4ß4-nAChR (Fig. 6C and D, Table 2). As DHßE does, mecamylamine, D-tubocurarine and methyllycaconitine block nicotine-induced peak currents mediated through 4ß2- or 4ß4-nAChR, with largely similar inhibitory potencies (Table 2). Hexamethonium more potently blocks 4ß2- than 4ß4-nAChR function (Table 2). Rank-order inhibition potency (IC₅₀ values and 95% confidence intervals provided in parentheses) is DHßE (0.89 µM; 0.72–1.10) > > mecamylamine (4.2 µM; 3.5–5.1) >> hexamethonium (30 µM; 21–42) methyllycaconitine (35 µM; 31–38) = D-tubocurarine (35 µM; 26–46) for 4ß2-nAChR and DHßE (1.2 µM; 0.93–1.50) >> mecamylamine (4.9 µM; 3.2–7.4) > > methyllycaconitine (26 µM; 17–39) d-tubocurarine (50 µM; 37–68) > hexamethonium (91 µM; 74–110) for 4ß4-nAChR.

View larger version (25K): [in this window] [in a new window]   Figure 6.  Antagonism of 4ß2- and 4ß4-nAChR function 3 µM (A) or 1 µM (B) nicotine (EC50 concentration)-induced whole-cell currents obtained from exposure to SH-EP1-h4ß2 (left) or -h4ß4 (right) cells alone or in the presence of different concentrations of DHßE are superimposed. C and D, effects of coexposure to DHßE (, ), mecamylamine (MEC; , ), hexamethonium (HEXA; •, ), D-tubocurarine (d-TC; , ) or methyllycaconitine (MLA; , ) on nicotine-evoked whole-cell peak current responses of 4ß2- (3 µM nicotine, C) or 4ß4-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.

View this table: [in this window] [in a new window]   Table 2.  Comparison of antagonist properties of 4ß2- and 4ß4-nAChR

View larger version (29K): [in this window] [in a new window]   Figure 7.  Comparison of antagonist action at 4ß2- and 4ß4-nAChR Nicotine concentration–response curves alone or in the presence of 0.3 µM DHßE (Aa, 4ß2-nAChR; Ab, 4ß4-nAChR), 1 µM MEC (Ba, 4ß2-nAChR; Bb, 4ß4-nAChR) or 0.5 µM HEXA (Ca, 4ß2-nAChR; Cb, 4ß4-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.

Current–voltage relationships for 4ß2- and 4ß4-nAChR

Whole-cell current traces recorded using K⁺ electrodes and obtained for 100 µM nicotine-induced activation of 4ß2-nAChR after stepwise changes in V_H prior to agonist application show full inward rectification at positive V_H (Fig. 8Aa), but when using the same protocol for 4ß4-nAChR responses, inward rectification at positive V_H was substantial but incomplete (Fig. 8Ab). Results compiled from studies using six cells confirm that 4ß4-nAChR-mediated whole-cell currents exhibit less inward rectification than 4ß2-nAChR-mediated currents, suggesting roles for ß subunits in the phenomenon (Fig. 8Ac). In additional studies using pipette solutions containing 30 mM NaCl to enhance outward currents, responses to 1 mM ACh at V_H values between –80 and +100 mV were recorded for 4ß2- (Fig. 8Ba) and 4ß4-nAChR (Fig. 8Bb). Figure 8Bc summarizes the current–voltage relationship curves (from –80 to +100 mV) for 4ß2- and 4ß4-nAChR from six cells tested, and confirms weaker inward rectification for 4ß4-nAChR- than for 4ß2-nAChR-mediated whole-cell currents.

View larger version (21K): [in this window] [in a new window]   Figure 8.  Current-voltage (I–V) relationships for 4ß2- and 4ß4-nAChR A, whole-cell peak currents evoked by 100 µM nicotine (indicated by black horizontal bar) at different VH values (–80, 0, +60 mV) are shown above current–voltage response curves normalized to the response to nicotine at –100 mV (*) for 4ß2- (Aa; in Ac) or 4ß4- (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 4ß2- (a) and 4ß4- (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 VH values (–80, 0, +80 mV) are shown above current–voltage response curves normalized to the response to nicotine at –80 mV (*) for 4ß2- (Ba; in (Bc) or 4ß4- (Bb; • in Bc) nAChR.

	Discussion

Top Abstract Introduction Methods Results Discussion References

ß subunits influence sensitivity of 4*-nAChR to functional inactivation

Given that nAChR appear to undergo more than one phase of functional inactivation, we propose the use of at least two terms to describe these processes and refined approaches to tease them apart. ‘Acute desensitization’ is defined in the present study as it was by Ochoa et al. (1990): the loss in nAChR response during a single stimulus or exposure to agonist, reflecting decay of inward currents typically recorded from a cholinoceptive cell from a peak to a lower, steady-state level. Levels of acute desensitization can be quantified in terms of time or rate constants of the decay in response and by the ratio between steady-state and peak inward current responses. Provided that the duration of agonist exposure is limited, and that the time between agonist challenges is adequately long, full recovery should occur from acute desensitization within seconds to minutes. ‘Functional rundown’ is defined as the reduction in peak responses to agonist due to repetitive agonist exposures. Ochoa et al. (1990) provided an illustration (see their Fig. 3B), but not a specific definition, of functional rundown. By our definition, functional rundown can be quantified in terms of peak current amplitude either as a function of cumulative time of repetitive agonist exposures or of numbers of agonist exposures.

The current study addresses the roles of ß subunits in 4*-nAChR acute desensitization and functional rundown. They show dramatically lower acute desensitization and functional rundown of 4ß4-nAChR compared to 4ß2-nAChR. The high conservation of amino acids across human ß2 and ß4 subunits in putative transmembrane domains, including the M2 domain thought to line the channel itself, suggests that alternative sites may be involved in differences in functional inactivation. One candidate is the second major cytoplasmic loop, changes in which influence nAChR acute desensitization (Kuo et al. 2005). The finding that there is slower desensitization of 4ß4-nAChR than of 4ß2-nAChR may have physiological and pathophysiological significance. For example, given their comparable sensitivity to nicotine, 4ß4-nAChR would be more resistant to functional inactivation by nicotine than 4ß2-nAChR (see Gentry et al. 2003), meaning they could play an important role in maintaining cholinergic network activity after chronic exposure to smoking-relevant nicotinic concentrations (100–500 nM), under conditions where other nAChR are either deeply desensitized (Pidoplichko et al. 1997) or not activated (Zhao et al. 2003). Consistent with such a perspective, ß4-subunit knockout mice have altered nicotine withdrawal symptoms (Salas et al. 2004). Diversity in nAChR dependent on whether they contain ß2 or ß4 subunits (perhaps in combination with additional subunits from the family) thus could allow for different physiological outcomes relevant to nicotine reinforcement, dependence and withdrawal.

ß subunits influence 4*-nAChR pharmacology

Many studies have indicated that mammalian brain 4ß2-nAChR are the nAChR subtype with the highest binding affinity for nicotine (Benowitz et al. 1989; Hsu et al. 1995; Fenster et al. 1997; Lukas et al. 1999; Jensen et al. 2005; Lukas, 2006). In studies using nAChR heterologously expressed in the oocyte system, the binding affinity of ACh for rat 4ß2-nAChR was 2-fold higher than for rat 4ß4-nAChR (Parker et al. 1998). However, patch-clamp whole-cell recording using the same expression system showed a 2-fold higher functional agonist potency for ACh acting at 4ß4-nAChR compared to 4ß2-nAChR (Francis et al. 2000). Our results show that nicotine, epibatidine and ACh have 2.4–3.4-fold higher functional potency when acting at 4ß4-nAChR than at 4ß2-nAChR. Moreover, 4ß4-nAChR have higher affinity than 4ß2-nAChR for cytisine and lobeline, both of which are fully efficacious at 4ß4-nAChR but are only partial agonists at 4ß2-nAChR. Interestingly, radioligand binding studies indicate that human 4ß2-nAChR have higher agonist-binding affinities than human 4ß4-nAChR (Eaton et al. 2000), but this simply suggests that functional and binding assays may assess ligand interactions with different states (resting, desensitized) of the receptor. Thus, 4ß2-nAChR remain as the highest affinity binding sites for nicotinic agonists when assessed using radioligand binding, but 4ß4-nAChR have higher agonist affinities when surveyed using whole-cell (or ion flux; Eaton et al. 2000) assays.

Antagonist sensitivities of the two 4*-nAChR subtypes studied are similar for the agents tested except for hexamethonium, which engages in non-competitive block of 4ß2-nAChR, but has a competitive and weaker inhibitor profile when acting at 4ß4-nAChR. A caveat is that these experiments assessed the effects on whole-cell peak currents, which may or may not be the most important indicator of physiological or pharmacological status of nAChR in vivo. Nevertheless, from a potential therapeutic perspective, these studies suggest that nicotinic agonists selectively targeting 4ß4-nAChR could be designed that would be much less efficacious and potent when acting at 4ß2-nAChR, and perhaps diseases affecting nicotinic signalling and elements of nicotine dependence could be treated with better outcome by targeting 4ß4-nAChR.

ß subunits influence 4*-nAChR inward rectification

Inward rectification is an important functional feature of several ligand- and voltage-gated ion channels, including 4*-nAChR (Haghighi & Cooper, 1998), but the mechanisms involved remain unclear. It has been suggested that polyamine interactions with a subsite in the channel-lining domain of nAChR blocks the channel pore in a voltage-dependent manner, leading to receptor inward rectification (Haghighi & Cooper, 1998, 2000). Apparent inverse relationships between inward rectification and Ca²⁺ permeability have been suggested in other studies (Lewis, 1979; Adams et al. 1980; Hume et al. 1991; Verdoorn et al. 1991; Dingledine et al. 1999). Other models consider relationships between roles for intracellular K⁺ in destabilization of polyamines with nAChR during depolarization and/or in outward ion flow (Washburn et al. 1997; Haghighi & Cooper, 1998). We have found that heterologously expressed, human 4ß4-nAChR show less inward rectification than 4ß2-nAChR, even when the intracellular Na⁺ concentration is high. There is a previous report of lower Ca²⁺ permeability in ß4-containing than in ß2-containing nAChR (Haghighi & Cooper, 1998). However, further studies comparing 4ß2- and 4ß4-nAChR have promise with respect to elucidation of mechanisms engaged in inward rectification.

Potential physiological relevance of ß4-containing nAChR in the brain

nAChR ß4 subunits play clear roles as assembly partners with 3 subunits in 3ß4*-nAChR that mediate trans-ganglionic signalling (Duvoisin et al. 1989; Rust et al. 1994; Xu et al. 1999). ß4 subunits also are expressed in the central nervous system as both mRNA and protein (Dineley-Miller & Patrick, 1992; Tarroni et al. 1992; Poth et al. 1997; Quick et al. 1999; Quik et al. 2000; Klink et al. 2001; Gahring et al. 2004). In several brain regions, including the basal ganglia, cerebellum, hippocampus and cortex, the ß4 subunit is a candidate assembly partner for the 4 subunit because 3 subunits are either absent or expressed at low levels (Dineley-Miller & Patrick, 1992). ß4 subunits are prominently expressed in thalamic somatosensory relay nuclei and in somatosensory cortex (Gahring et al. 2004), suggesting involvement in sensory processing. ß4-null mice have altered responses in anxiety-related tests (Salas et al. 2003) and upon nicotine withdrawal (Salas et al. 2004), and have higher resistance to nicotine-induced seizures (Kedmi et al. 2004). Dopamine neurons freshly dissociated from the rat ventral tegmental area express functional nAChR with properties suggestive of the presence of ß4 subunits that are expressed as mRNA in the same region (Wu et al. 2005). These putative ß4*-nAChR mediate whole-cell current responses to ACh that are of higher amplitude (400–1000 pA) and desensitize more slowly than 4ß2-nAChR-mediated responses. These ß4*-nAChR are sensitive to the nicotinic agonist cytisine, but insensitive to the 4ß2-nAChR-selective agonist RJR-2403 (Papke et al. 2000). In addition, these ß4*-nAChR are highly sensitive to the nicotinic receptor antagonist mecamylamine, but show low sensitivity to the 4ß2-nAChR-selective antagonist DHßE. While the possibility is that these nAChR have a subunit composition more complicated than 4 plus ß4, the similarities between their properties and those of 4ß4-nAChR defined in the current study are striking, and involvement of ß4 subunits seems clear. Functional ß4*-nAChR in midbrain dopamine neurons may contribute to nicotine-induced modulation of midbrain dopamine neuronal function.

In summary, human 4ß2- and 4ß4-nAChR heterologously expressed in human SH-EP1 cells exhibit distinctive physiological and pharmacological properties. Heterologous expression of these entities and identification of ways to distinguish them provide insights into roles of these two 4*-nAChR subtypes in health and disease and as therapeutic targets.

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