Regions of β4·β2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors

Abstract

"Divisian of Chemi~'uy mtd nDit'isin. ~f Bialagv 156.29, Califaroir, i, stintW of TechnnleJll.v, Pasadena. CA 9/125, USA gc~ivcd 26 June 1992 Fifteen chimeric nicotinic receptor ,6 subunits were constructed consistinM of N-terminal neuronal ~4 sequences and C-terminal ~ sequanc¢~.flcsponscs to c~'tisin¢, nicotine, or tot ramcthylammonium wcrt compared to acetylcholine rcspo::scs {'or these subunits cxpre,,~l in Xenapu.s oocytes with =3 subunits, The results show that (i) two residues in the ¢xtracellular domain of chimeric.~4,j~2 subunits (IOII,~2FIfl4V, 110.~.S/,~4T) acamunt for much of the relative cytisine sensitivity; and (it) four extr'acellular residu~ ofehimeric,64,J12 subunits (112~2A/,~4V. 1 ! ~l.~.V/fl4l and I 15,P,.$/.a4R, 116/J2YI/~4S) account for most of th." relative tetramcthyl:tmmonium sensitivity. The ~ata did not r~rmit localization of nicotine ~flsitivity to any ~trticular r¢ilion. shows that both subunits contribute to the relative sensitivity to ganglionic stimulants and ncurotoxins [1.2], channel conductance and gating p+'operties [3-5] of nettronal nAChR's. Receptors containing the f14 subtype generally give larger rcsponst;s than receptors containing the ~2 subtype to ganglionic stimulants such as cytisine (CYT) and nicotine (NIC) [2]. To localize the regions of~4 and f12 that contribute to agonist selectivity, we constructed chimeras of~'4 and ~2 consisting of N.terminal sequences from ,84 varying length and appropriate C-terminal counterparts from f12. These were expressed in combination with ct3 in Xenopus oocytes. 2, MATERIALS AND METHODSFifteen f14,,f12 chimeras were constructed using a previously de. scribed PCR method [6] so that they cont~.ined an N-terminal end from.t/4 and a C-terminal end from B2, For example, the arrow labeled ? in Fig, 1 denotes the chimera/24(?).,~2 that comains the ? most N-terminal residues from/'/4 and the remaininll 4?0 C-terminal rest. dues tram B2. mRNA for a3 and the fl subunits wa~ transcribed in vitro [9] and L8 ng of each subunit was coinjected into stage V or VI Xen.pus laevis oocytes, The oocytes were incubated 2-7 days in a modified Barth's solution containing 5% horse.serum. We measured the peak current produced by bath application of30,uM acetylcholin¢ (ACh), 30 #aM cytisin¢ (CY'I'), 30/JNI nicotine (NIC), and 100/JM Correspondence address: H,A, l.,¢~tcr, Division of Biology 156-29.Pasadena, CA 91 I25, USA. Fax: (1) (tile) 564 8709, tetrumethylammonium (TMA) at u typi~l holdinll potential of -ISO mY usinll ;s two.electrode voltall¢ clamp, Receptors that lave ACh r~ponscs too larl~¢ for accurate rccordinli at -80 mV wcrt m~sured at more depolarized potentials (-70 to -~0 nzV), ACh responsms ofthe chimeric and wild-tyl~ (WT) rc~ptors were typically in tfl~ 100-2,000 nA ranlle. The recordinil solution contained 96 mM NaCI. 2 mM NaOH. I mM MgCI.., and S mM HEPF..5 (pH 7,4), External Ca"" was omitted to minimize the activation of the Ca =-activated CI" conduct. ance [8]. AlL recordings wart mad© at ambient tcmpcraturt (23-25'C1, 3, RESULTSThe ...

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

“…On the basis that Campos-Caro et al (46) have shown that there is no ␤2 subunit expressed in bovine chromaffin cells (which contain neuronal ␣3, ␣5, ␣7, and ␤4 subunits) it appears that EpI targets bovine ␣3␤4 receptors. However, there is pharmacological evidence that ␣3␤4 in bovine chromaffin cells behaves similarly to ␣3␤2 in the rat (46,50).…”

“…D, dose-dependent inhibition of ACh-evoked peak current by EpI (E) and [Tyr 15 ]EpI (q) normalized to the peak current amplitude of the AChevoked current obtained at -60 mV in the absence of EpI. The dose-response relations were fit with a single site adsorption isotherm giving IC 50 …”

α-Conotoxin BuIA, a Novel Peptide from Conus bullatus, Distinguishes among Neuronal Nicotinic Acetylcholine Receptors

“…If ␣-conotoxin BuIA blocks by binding to the subunit interface, we feel it likely that the toxin would have slow off-rate kinetics for receptors containing a ␤4 subunit at the ligand binding interface. The binding of other ligands is known to interact with nAChR ␣ and ␤ subunits, and nAChR subunit chimeras have been used to localize the residues that contribute to subunit-dependent ligand selectivity (34). The agonist cytisine selectively activates rat ␣x␤4 versus ␣x␤2 nAChRs expressed in Xenopus oocytes (35).…”

A Polymorphism in the Mouse Neuronal α4 Nicotinic Receptor Subunit Results in An Alteration in Receptor Function

“…Studies using nAChR subunit chimeras have identified potential roles for the extracellular domain in regulating sensitivity to agonists (Figl et al, 1992;Luetje et al, 1993;Corringer et al, 1998) and antagonists (Harvey et al, 1996), but this approach has not yielded much information about the role of the cytoplasmic loop in regulating neuronal nAChR function. One exception to this is a study done by Gross et al (1991), who detected a potential function for the cytoplasmic loop in a study that used ␣4/␣3 (residues 1-200 of ␣4 and 196 -474 of ␣3) and ␣3/␣4 (residues 1-195 of ␣3 and 201-599 of ␣4) chimeras.…”