The guanine nucleotide-binding protein, Gi, which inhibits adenylyl cyclase, has recently been shown to have three subtypes of the alpha-subunit, termed Gi alpha-1, Gi alpha-2 and Gi alpha-3. They share 87-94% amino-acid sequence homology and so are difficult to separate from one another. Among other functions, purified preparations activate K+ channels but there is confusion over which of the subtypes activates the muscarinic K+ channels of the atrial muscle of the heart: Gi alpha-3, also termed Gk, has been shown to activate this channel but it is not clear whether Gi alpha-1 does or does not. To clarify this problem, we expressed the subtypes separately in Escherichia coli to eliminate contamination by other subtypes and tested the recombinant alpha- chains on atrial muscarinic K+ channels. Although we anticipated that only Gi alpha-3 would have Gk activity, to our surprise all three recombinant subtypes were active, from which we deduce that the Gi subtypes are multifunctional.

A B S TRACT Arrival of agonist is generally thought to initiate the signal transduction process in G protein-receptor coupled systems. However, the muscarinic atrial K + (K+[ACh]) channel opens spontaneously in the absence of applied agonist, giving a noisy appearance to the current records. We investigated the nature and origin of the noise by measuring single channel currents in cell-attached or excised, insideout membrane patches. Guanosine triphosphate (GTP) produced identical single channel currents in a concentration-and Mg~+-dependent manner in the presence or absence of carbachol, but the requirements for GTP were greater in the absence of agonist. Hence the agonist-independent currents appeared to be produced by an endogenous G protein, Gk. This prediction was confirmed when an afffinity-purified, sequence-specific G~-3a antibody or pertussis toxin (PTX) blocked the agonistindependent currents. Candidate endogenous agonists were ruled out by the lack of effect of their corresponding antagonists. Thus agonist-independent currents had the same nature as agonist-dependent K+[ACh] currents and seemed to originate in the same way. We have developed a hypothesis in which agonist-free, empty receptors prime Gk with GTP and G k activates atrial K ÷ [ACh] channels producing basal currents or noise. Agonist-independent activation by G proteins of effectors including ion channels appears to be a common occurrence.

1. Four questions raised by previous studies that had shown activation of K+ channels by alpha subunits of the type 3 Gi protein are addressed in the present communication: a) are K+ channels specific for one Gi? b) are there more ionic channels under direct G protein control? c) can we confirm using recombinant G alpha s the results obtained with biochemically resolved G alpha s and continue ascribing the regulatory effector to this part of the alpha beta gamma holo‐G protein? and d) can we confirm that a single G alpha, Gs alpha in this case, is able to affect more than one type of effector function? 2. We found Gi alpha s are isoforms, that there exist also Gi‐insensitive, Go‐responsive K+ channels and that G alpha s can be multifunctional. Thus, a single receptor will elicit cellular responses that will depend on the endogenous G protein as well as the type of effector function expressed in it. 3. In another set of experiments we found that G beta gamma s, be they derived from human erythrocytes, human placenta, bovine brain or bovine retina, all inhibit Gk‐gated K+ channel activity as seen in inside out membrane patches with GTP as the driving nucleotide. In addition we noted that inhibition was much more effective under basal (no agonist in the pipette) than agonist stimulated conditions, as reported in earlier experiments in which beta‐adrenoceptors, Gs and catalytic unit of adenylyl cyclase had been incorporated into phospholipid vesicles. 4. We propose that one of the roles of G beta gamma s in membranes is to quench ligand independent G protein activation by unoccupied receptors. Other roles of G beta gamma s are: a) by re‐associating with GDP‐G alpha s, to promote interaction with receptors, and b) by dissociating from activated R.G alpha *GTP.beta gamma, to allow for receptor dissociation from GTP‐activated G alpha s, which is required to satisfy the catalytic mode of receptor action.

Heterotrimeric G3 proteins are though to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K+ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the alpha subunits of G proteins directly couple receptors to ionic channels, particularly K+ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K+ channel effectors.

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