GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα i3 decreases the basal activity (I basal ) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive' mutant of Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal and restored the ability of Gβγ to activate GIRK1 * , like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1 * . These results demonstrate that GIRK1 containing channels are regulated by both Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .

1. A combined biochemical and electrophysiological approach was used to determine the mechanism by which the auxiliary subunits of Ca2+ channel enhance the macroscopic Ca2+ currents. Xenopus oocytes were injected with RNA of the main pore-forming subunit (cardiac: alC), and various combinations of RNAs of the auxiliary subunits (a2/4 and ,82A).2. The single channel open probability (P.; measured at 0 mV) was increased -3-, -8-and

In the heart, Ca 2ϩ current via the voltage-dependent L-type channels (dihydropyridine-sensitive) underlies the plateau of the action potential and provides calcium ions necessary for initiation of cardiac cell contraction (2). Similar channels are found in smooth muscle, where they play a major role in regulation of tonus and contraction (3, 4), and in the nervous system (5, 6). L-type channels are composed of the following three subunits: the main, pore-forming ␣ 1C , the cytosolic ␤2, and the ␣ 2 ␦ subunit which is mostly extracellular (5, 7-11). ␣ 1C contains four homologous membrane domains numbered I-IV, each one with six transmembrane segments and a re-entrant P-loop that forms the pore lining; N-and C-terminal domains and the linkers connecting the domains I-II, II-II, and II-IV are cytosolic (see Ref. 7 for review, and see Fig. 6A for a scheme). The C terminus was implicated in Ca 2ϩ -and voltagedependent inactivation (12-15) and modulation by protein kinase A (16 -19); linker I-II contains the binding site for the ␤ subunit (20, 21).Cardiac and smooth muscle L-type channels are tightly regulated by hormonal and neuronal signals via G proteins and protein kinases (22,23). Protein kinase C (PKC) 1 is one of such regulators; its actions appear to be tissue-and species-specific. PKC activators, such as phorbol esters and diacylglycerols, increase Ca 2ϩ channel currents in cardiac and smooth muscle cells of various mammals (24 -33), and PKC has been implicated in mediating the stimulation of Ca 2ϩ channels by intracellular ATP (34), angiotensin II (26), glucocorticoids (28), pituitary adenylate cyclase-activating polypeptide (33), and arginine-vasopressin (32). PKC up-regulation results from changes in channel gating because it is accompanied by an increase in single channel open probability, P o (30,35,36). In many cases, a biphasic effect of PKC activators has been described, with an increase followed by a later decrease (25,27,30), and some preparations such as adult guinea pig heart cells (37, 38) respond to phorbol esters only by a decrease in Ca 2ϩ currents, an effect that may not be mediated by PKC (38). The biphasic response to PKC stimulators is fully reconstituted when expression of L-type channels in Xenopus oocytes is directed by RNA extracted from rat heart (39, 40) or cRNA of rabbit cardiac ␣ 1C subunit (39). Increase of Ca 2ϩ channel activity by phorbol esters has also been observed in a mammalian cell line (baby hamster kidney) expressing the rabbit cardiac ␣ 1C (36). The potentiation by phorbol esters of Ca 2ϩ channels expressed in the oocytes is mediated by PKC because it is mimicked by diacylglycerols and blocked by specific PKC inhibitors (39,40).Both ␣ 1C and ␤ are substrates for PKC-catalyzed phosphorylation (Ref. 41 and references therein). ␣ 1C subunit has been recognized as the target for the Ca 2ϩ channel enhancement caused by PKC, since coexpression of the auxiliary subunits was not necessary to reproduce the effect of phorbol esters; on the contrary, coexpression of the ␤ subunit...

Neuronal voltage-dependent Ca2؉ channels of the N (␣ 1B ) and P/Q (␣ 1A ) type are inhibited by neurotransmitters that activate G i/o G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of G␤␥ subunit of G proteins to the channel. Since cardiac and neuronal L-type (␣ 1C ) voltage-dependent Ca 2؉ channels are not modulated in this way, they are presumed to lack interaction with G␤␥. However, here we demonstrate that both G␤␥ and calmodulin directly bind to cytosolic N and C termini of the ␣ 1C subunit. Coexpression of G␤␥ reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca 2؉ , and is eliminated by EGTA. The N and C termini of ␣ 1C appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of G␤␥. Deletion of the N terminus profoundly impairs the Ca 2؉ /calmodulin-dependent inactivation. We propose that G␤␥ and calmodulin regulate the L-type Ca 2؉ channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of ␣ 1C .Voltage-dependent Ca 2ϩ channels (VDCCs) 1 are crucial for neuronal and muscular excitability (1). Mammalian VDCCs fall into several families distinguished by pharmacological and biophysical properties (L, N, P/Q, T, and R type) and the molecular identity of the main, poreϪforming subunit, ␣ 1 (2-4). The neuronal N-and P/Q-type channels, based on ␣ 1B and ␣ 1A , respectively, are crucial for neurotransmitter release (3). Ltype Ca 2ϩ channels containing the "cardiac-type" ␣ 1C subunit regulate contraction of cardiac and smooth muscle, and excitability and gene expression in the brain (2, 5, 6). The ␣ 1 subunits contain four homologous membrane domains numbered IϪIV and 5 large intracellular segments: N terminus (NT), C terminus (CT), and linkers between the domains (often called loops L 1 , L 2 , and L 3 ). There is also a large number of short intracellular linkers between transmembrane segments within each domain.Activation in all voltage-dependent channels is initiated by a voltage-driven shift in charged transmembrane elements (7). Nevertheless, the parts of the channel and the auxiliary subunits which are not exposed to the membrane electrical field may substantially modulate the gating (for reviews related to Ca 2ϩ channels, see Ref.3). In particular, VDCCs are strongly and specifically modulated by neurotransmitters acting via heterotrimeric G proteins, via actions on the cytosolic parts of the channel. Some of the modulations are mediated by G protein-triggered second messenger cascades, often via protein kinases A and C (PKA and PKC, respectively), others by a direct interaction with G protein subunits (1, 8 -14). Both PKC and PKA alter VDCC gating parameters acting via cytosolic parts of ␣ 1 or via the ancillary ␤ subunit (15-19).Neuronal VDCCs are usually inhibited by G protein-coupled rece...