Human α4β2 Neuronal Nicotinic Acetylcholine Receptor in HEK 293 Cells: A Patch-Clamp Study

Abstract

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, alpha4 and beta2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 microM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha4beta2 receptors is (-)-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 >> (-)-nicotine > ACh >> (-)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 microM ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nM and 1.5 microM. The current-voltage relationship of human alpha4beta2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha4beta2 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca 2+ -binding sites

“…In contrast, the receptors (␣4) 2 (␤2) 3 and (␣1) 2 ␤1␥␦ would possess two sites with two acid residues and a serine, which in other systems have been shown to chelate calcium ions (for instance in synaptotagmin). This difference would explain the potentiation by calcium in (␣7) 5 (57) much stronger than in (␣4) 2 (␤2) 3 (59) and (␣1) 2 ␤1␥␦ (56).…”

Four Excitatory Postsynaptic Ionotropic Receptors Coactivated at the Motoneuron–Renshaw Cell Synapse

“…The MLA-sensitive component has the characteristics of a response mediated by a homomeric ␣ 7 receptor: it is very sensitive to MLA (Alkondon et al, 1992;Buisson et al, 1996;ChavezNoriega et al, 1997). Its rapid decay may reflect fast dissociation from the ␣ 7 receptor (EC 50 values Ͼ100 M) (Couturier et al, 1990;Alkondon et al, 1992; Chavez-Noriega et al, 1997) or, Figure 6.…”

“…This new hypothesis implied that extrasynaptic receptors are activated by a low concentration of ACh, which was difficult to reconcile with the low affinity of the known nAChRs. However, it was recently reported that some recombinant nAChRs have apparent dissociation constants for ACh in the low micromolar range (Buisson et al, 1996;Zwart et al, 1998;Covernton and Connolly, 2000;Moroni et al, 2006). The slow dissociation of ACh from such receptors, if they were present on the RCs, could explain the long duration of the RC synaptic current.…”

Cholinergic drugs potentiate human nicotinic α4β2 acetylcholine receptors by a competitive mechanism

“…Research into function and pharmacology of human a4h2 nicotinic acetylcholine receptors has been fostered by the availability of systems for the selective expression of specific subtypes of nicotinic acetylcholine receptors, such as Xenopus laevis oocytes Chavez-Noriega et al, 1997), human embryonic kidney (HEK) cells Buisson et al, 1996;Chavez-Noriega et al, 2000), and, more recently, human epithelial SH-EP1 cells (Eaton et al, 2003). Agonists of the nicotinic acetylcholine receptor interact with the agonist recognition sites of these receptors to induce conformational changes in the membrane protein, which lead to ion channel opening and receptor desensitization.…”