Expression of functional, recombinant ␣7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor ␣7 subunit. In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding. Upon application of 1 mM nicotine, currents were undetectable in HEK293 cells expressing only the ␣7 subunit. In contrast, co-expression of ␣7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous ␣7 receptors. Immunoprecipitation studies demonstrate that ␣7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of ␣7 protein on the plasma membrane of cells lacking ric-3, but surface ␣-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of ␣7 receptors in HEK293 cells. Nicotinic acetylcholine receptors (nAChRs)1 are members of the neurotransmitter-gated ion channel superfamily. They are widely expressed in the central and peripheral nervous system (1) where they influence numerous cellular and physiological processes. At least 17 different genes that code for nAChR subunits have been identified (2, 3), and they assemble as pentamers in different combinations to form a diverse set of nAChR subtypes (4, 5). The simplest case is the homopentameric complex such as that formed by the nAChR ␣7 subunit. The ␣7 receptor, for which ␣-bungarotoxin (␣-Bgt) is a specific and high affinity antagonist, is one of the most abundant receptor subtypes in the mammalian brain (6, 7). The high Ca 2ϩ permeability of the ␣7 receptor (8) suggests an involvement in the activation of Ca 2ϩ -dependent events in neurons such as transmitter release, participation in signal transduction, and a variety of modulatory effects (9). In addition, ␣7 receptors have been implicated in a number of diseases such as schizophrenia, Alzheimers, and Parkinsons disease (1, 10 -12).Heterologous expression of the ␣7 subunit in Xenopus oocytes results in homooligomeric, ␣-Bgt-sensitive receptors that activate and inactivate quickly and are highly permeable to Ca 2ϩ (8,13,14), similar to the properties of ␣7 nAChRs in neuronal cells. Although there have been reports of successful functional expression in some mammalian cell lines (15-18), measurable levels of functional receptors have been difficult to achieve in multiple cell types and this phenomenon appears to be host-cell dependent (19). The reasons for poor heterologous surface expression in these cells are not well understood. Strategies to increase the number of functional receptors on the cell...

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.

The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the mu-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA mu-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.