␣7-Nicotinic acetylcholine receptors (␣7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human ␣7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human ␣7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional ␣7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human ␣7-nAChRs. Currentvoltage relationships show inward rectification for agonist-induced currents. Human ␣7-nAChRs exhibit some sensitivity to ␣7-nAChR antagonists ␣-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and ␣7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of ␣7-nAChR function. Whole-cell current peak amplitudes and halftimes for inactivation of ␣7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca 2ϩ , suggestive of high Ca 2ϩ permeability of the ␣7-nAChR channel. Thus, heterologously expressed human ␣7-nAChR in mammalian cells have properties of native ␣7-nAChR or of ␣7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of ␣7-nAChR function.

The purpose of these experiments was to examine the role of the human cerebellum in the acquisition and retention of conditioned reflexes. Normal human subjects and patients with cerebellar lesions were tested for their capacity to acquire, retain and express conditioned eyeblink responses. In acquisition tests, subjects were trained in a delay classical conditioning paradigm using a tone conditioned stimulus and a midline forehead tap as an unconditioned stimulus. While normal subjects developed anticipatory eyeblinks to the tone in one session, patients with cerebellar lesions failed to acquire conditioned responses in four consecutive training sessions. The conditioning deficit was bilateral even in patients with a unilateral cerebellar pathology. The same groups of subjects were tested for the presence of eyeblinks to a visual threat. In these experiments, both normal subjects and patients with cerebellar lesions exhibited a high level of responding when they saw an object approaching their face. These eyeblinks to the visual threat are probably naturally acquired conditioned responses because they extinguish in normal subjects if they are not reinforced by the unconditioned cutaneous stimulus. In addition, the stimulus of seeing an approaching object blocks the acquisition of classically conditioned eyeblinks to a new conditioned stimulus in normal subjects. These data imply that patients with cerebellar lesions who cannot acquire new classically conditioned responses are able to retain and express conditioned eyeblinks which were acquired before the onset of the pathology. Consequently, cerebellum-dependent neural substrates which are involved in learning new conditioned reflexes do not seem to be required for the storage of naturally learned conditioned responses.

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To evaluate possible physiological roles of the large cytoplasmic loops (C2) and neighboring transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits, we generated novel fusion constructs in which human nAChR ␣4, ␤2, or ␤4 subunit C2 or C2 and neighboring sequences were replaced by corresponding sequences from the mouse serotonin type 3A (5-HT 3A ) receptor subunit. Following stable expression in human SH-EP1 cells, we found that extensive sequence substitutions involving third and fourth transmembrane domains and neighboring "proximal" C2 sequences (e.g., ␤2 H322-V335 and V449-R460) did not allow functional expression of nAChR containing chimeric subunits. However, expression of functional nAChR was achieved containing wild-type ␣4 subunits and chimeric ␤2 (␤2) subunits whose "nested" C2 domain sequences K336-S448 were replaced with the corresponding 5-HT 3A subunit sequences. Whereas these findings suggested indispensable roles for M3/M4 transmembrane and/or proximal C2 sequences in ␣4␤2-nAChR function, nested C2 sequences in the ␤2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of ␣4␤2-nAChR compared with ␣4␤2-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of ␣4␤2 function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of ␣4␤2-nAChR than for ␣4␤2-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.Nicotinic acetylcholine receptors (nAChR) are members of a ligand-gated ion channel superfamily, each comprised of a homo-or heteropentameric assembly of distinct subunits (Lindstrom, 1996;Lukas, 1998;Karlin, 2002). All nAChR subtypes mediate transmembrane ion flux upon activation by interaction with the endogenous neurotransmitter acetylcholine (ACh) or the tobacco alkaloid nicotine. However, each nAChR subtype possesses unique channel properties dictated in part by the subtle diversity of its constituent subunits.Each of the 17, genetically distinct, vertebrate nAChR subunits identified to date share a common topology containing a large extracellular N-terminal domain, four transmembrane domains, a short cytoplasmic domain between the first and second transmembrane segments, a short extracellular domain between second and third transmembrane segments, a large second cytoplasmic loop (C2) situated between the third (M3) and fourth (M4) transmembrane domains, and a short C-terminal extracellular tail. The N-terminal domain contains key elements for ligand-binding/recognition (Sine, 2002), and the transmembrane domains anchor the proteins in the plasma membrane and contribute to channel kinetics and ion selectivity (Corringer et al., 2000). These structural domains are well conserved among different subunits and have been studied extensively. On the other hand, the less studied C2 domain of each subunit contains unique sequence...

Heterologous expression of functional, nicotinic acetylcholine receptors (nAChR) in mammalian cells has been difficult to achieve or optimize, even for nAChR containing only one kind of subunit. In this study, we determined effects of lowered temperature or of exposure to the protein synthesis inhibitor cycloheximide (CHX) on cell surface expression of homomeric a7-nAChR in transfected SH-EP1 human epithelial cells. We found that incubation of cells for 2 days at 25°C or in the presence of 0.5-2 lg/mL of CHX caused four-or eight-fold increases, respectively, in surface binding sites for 125 I-labeled a-bungarotoxin (I-Bgt). These increases were accompanied by increases in peak whole-cell current responses to nicotinic agonists. Either treatment lowered protein synthesis and cell proliferation, but experiments using puromycin indicated that a reduction in protein synthesis or cell proliferation per se was not sufficient to increase surface binding. I-Bgt binding to whole-cell membrane pools increased in response to either treatment, suggesting that the increase in surface binding was due, at least in part, to an increase in intracellular receptor levels. The cyclophilin inhibitor cyclosporin A reduced surface expression in untreated as well as CHX-or 25°C-treated cells.The results suggest practical means for increasing cell surface and functional expression of a7-nAChR. Although these effects are not simply due to protein synthesis inhibition or reduced cell proliferation, they do involve an increase in intracellular receptor pool size.