Heart rate is slowed in part by acetylcholine-dependent activation of a cardiac potassium (K+) channel, IKACh. Activated muscarinic receptors stimulate IKACh via the G-protein beta gamma-subunits. It has been assumed that the inwardly rectifying K(+)-channel gene, GIRK1, alone encodes IKACh. It is now shown that IKACh is a heteromultimer of two distinct inwardly rectifying K(+)-channel subunits, GIRK1 and a newly cloned member of the family, CIR.

Homozygous weaver mice are profoundly ataxic because of the loss of granule cell neurons during cerebellar development. This granule cell loss appears to be caused by a genetic defect in the pore region (Gly156-->Ser) of the heterotrimeric guanine nucleotide-binding protein (G protein)-gated inwardly rectifying potassium (K+) channel subunit (GIRK2). A related subunit, GIRK1, associates with GIRK2 to constitute a neuronal G protein-gated inward rectifier K+ channel. The weaver allele of the GIRK2 subunit (wvGIRK2) caused loss of K+ selectivity when expressed either as wvGIRK2 homomultimers or as GIRK1-wvGIRK2 heteromultimers. The mutation also let to loss of sensitivity to G protein betagamma dimers. Expression of wvGIRK2 subunits let to increased cell death, presumably as a result of basal nonselective channel opening.

G protein-activated inwardly rectifying K ÷ channel subunits GIRK1 (Kit 3.1), GIRK2 (Kit 3.2), and CIR (Kir 3.4) were expressed individually or in combination in Xenopus oocytes and CHO cells. GIRKI coexpressed with CIR or GIRK2, produced currents up to 10-fold larger than any of the subunits expressed alone. No such clear synergistic effects were observed upon coexpression of CIR/GIRK2 under the same conditions. Coexpression of G protein i~/ (Gm~2) increased the current through GIRKllGIRK2 and GIRK2 channels. G~ subunits purified from bovine brain, increased channel activity 50-1000-fold in patches from cells expressing GIRKllGIRK2 or GIRK2 alone. The single GIRKIIGIRK2 channels resembled previously described neuronal G protein-gated K ÷ channels. In contrast, single GIRK2 channels were short-lived and unlike any previously described neuronal K ÷ channel. We propose that some neuronal G protein-activated inward rectifier K ÷ channels may be formed by a GIRKllGIRK2 heteromultimer and that G,~ activation may involve both subunits.

In locus coeruleus neurons, substance P (SP) suppresses an inwardly rectifying K+ current via a pertussis toxin-insensitive guanine nucleotide binding protein (G protein; G.0npTrx), whereas somatostatin (SOM) or [Met]enkephalin (MENK) enhances it via a pertussis toxin-sensitive G protein (Gprx). The interaction of the SP and the SOM (or MENK) effects was studied in cultured locus coeruleus neu-rons. In neurons loaded with guanosine 5'- [y-thio] GPTx also responds to a closing signal from GnonPTX. The closing signal is stronger than the opening signal.Inward rectifier K+ channels exist in various cell types and determine the resting conductance and potential (1-4). In addition to these ordinary inward rectifiers, there is another class of inward rectifiers, the guanine nucleotide binding protein (G protein)-coupled inward rectifiers. In atrial cells Gprotein regulation of inward rectifiers is responsible for the hyperpolarization caused by stimulation of the vagal nerve (5-8).G-protein-coupled inward rectifier K+ channels also exist in various types of vertebrate neurons, and the modulation of these channels generates slow synaptic potentials (9-16). In cholinergic neurons from the nucleus basalis of Meynert, substance P (SP) excites neurons by reducing an inward rectifier current (9), and this effect is mediated by a pertussis toxin-insensitive G protein (GnonPTx) (14). In MATERIALS AND METHODSA detailed description of the methods has been given (18). Neuronal cultures from the locus coeruleus were made as described (18,19). For electrophysiology, the tight seal wholecell patch clamp technique was used. The external solution (10 mM K+ solution) contained 141 mM NaCl, 10 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 11 mM D-glucose, 0.0005-0.001 mM tetrodotoxin, and -5 mM Hepes-NaOH (pH 7.4). The patch pipette solution contained 120 mM potassium aspartate, 40 mM NaCl, 3 mM MgCl2, 0.25 mM CaCl2, 0.5 mM EGTA-KOH, 2 mM Na2ATP, 0.1 mM Na3GTP (or 0. Fig. IA, application of MENK (1 ,uM) increased the membrane conductance. The average conductance increase was 14.7 + 2 nS (n = 25). Fig. lB shows MENK-induced currents, which were obtained by subtracting the control currents from the current during the effect of MENK As shown in Fig. 1C, the MENK-induced currents exhibited an inward rectification with a reversal potential near the K+ equilibrium potential (EK = -66 mV) in both GTPloaded cells (open circles) and GTP[yS]-loaded cells (solid circles). Thus, the properties of the MENK-induced conductance are essentially the same as those of the SOM-induced current (13).SP Effects. SP reduces a resting K+ conductance in nucleus basalis neurons, and the SP-suppressed conductance rectifies to the inward direction (9). Thus, the SP effect was an approximate mirror image of the SOM or MENK effects.The effects of SP on locus coeruleus neurons are more complicated than those on nucleus basalis neurons (18,20). As shown in Fig. 2A