Tatiana Ivanina

Gαi Controls the Gating of the G Protein-Activated K+ Channel, GIRK

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.

CA2+ CURRENT ENHANCEMENT BY ALPHA 2/DELTA AND BETA SUBUNITS IN XENOPUS OOCYTES : CONTRIBUTION OF CHANGES IN CHANNEL GATING AND ALPHA 1 PROTEIN LEVEL

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: alpha 1C), and various combinations of RNAs of the auxiliary subunits (alpha 2/delta and beta 2A). 2. The single channel open probability (Po; measured at 0 mV) was increased approximately 3‐, approximately 8‐ and approximately 35‐fold by alpha 2/delta, beta 2A and alpha 2/delta+beta 2A, respectively. The whole‐cell Ca2+ channel current was increased approximately 8‐ to 10‐fold by either alpha 2/delta or beta 2A, and synergistically > 100‐fold by alpha 2/delta+beta 2A. The amount of 35S‐labelled alpha 1 protein in the plasma membrane was not changed by coexpression of beta 2A, but was tripled by coexpression of alpha 2/delta (either with or without beta). 3. We conclude that the increase in macroscopic current by alpha 2/delta is equally due to changes in amount of alpha 1 in the plasma membrane and an increase in Po, whereas all of the effect of beta 2A is due to an increase in Po. The synergy between alpha 2/delta and beta in increasing the macroscopic current is due mainly to synergistic changes in channel gating.

Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

The role of the cytosolic N terminus of the main subunit (α1C) of cardiac L-type voltage-dependent Ca2+ channel was studied inXenopus oocyte expression system. Deletion of the initial 46 or 139 amino acids (a.a.) of rabbit heart α1C caused a 5–10-fold increase in the whole cell Ca2+ channel current carried by Ba2+ (IBa), as reported previously (Wei, X., Neely, A., Olcese, R., Lang, W., Stefani, E., and Birnbaumer, L. (1996) Recept. Channels 4, 205–215). The plasma membrane content of α1C protein, measured immunochemically, was not altered by the 46-a.a. deletion. Patch clamp recordings in the presence of a dihydropyridine agonist showed that this deletion causes a ∼10-fold increase in single channel open probability without changing channel density. Thus, the initial segment of the N terminus affects channel gating rather than expression. The increase in IBa caused by coexpression of the auxiliary β2A subunit was substantially stronger in channels with full-length α1C than in 46- or 139-a.a. truncated mutants, suggesting an interaction between β2A and N terminus. However, only the I–II domain linker of α1C, but not to N or C termini, bound β2A in vitro. The well documented increase of IBa caused by activation of protein kinase C (PKC) was fully eliminated by the 46-a.a. deletion. Thus, the N terminus of α1C plays a crucial role in channel gating and PKC modulation. We propose that PKC and β subunit enhance the activity of the channel in part by relieving an inhibitory control exerted by the N terminus. Since PKC up-regulation of L-type Ca2+ channels has been reported in many species, we predict that isoforms of α1C subunits containing the initial N-terminal 46 a.a. similar to those of the rabbit heart α1C are widespread in cardiac and smooth muscle cells.

The N terminus of the Cardiac L-type Ca2+ Channel α1C Subunit THE INITIAL SEGMENT IS UBIQUITOUS AND CRUCIAL FOR PROTEIN KINASE C MODULATION, BUT IS NOT DIRECTLY PHOSPHORYLATED

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca2+ channel α1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901–17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of α1Cleave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of α1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of α1C expressed inXenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathioneS-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.

Na+ Promotes the Dissociation between GαGDP and Gβγ, Activating G Protein-gated K+ Channels

G protein-gated K+channels (GIRK, or Kir3) are activated by the direct binding of Gβγ or of cytosolic Na+. Na+ activation is fast, Gβγ-independent, and probably via a direct, low affinity (EC50, 30–40 mm) binding of Na+ to the channel. Here we demonstrate that an increase in intracellular Na+ concentration, [Na+]in, within the physiological range (5–20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na+ effect. It is inhibited by a Gβγ scavenger, hence it is Gβγ-dependent; but it does not require GTP. We hypothesized that Na+ elevates the cellular concentration of free Gβγ by promoting the dissociation of the Gαβγ heterotrimer into free GαGDP and Gβγ. Direct biochemical measurements showed that Na+ causes a moderate decrease (∼2-fold) in the affinity of interaction between GαGDP and Gβγ. Furthermore, in accord with the predictions of our model, slow Na+ activation was enhanced by mild coexpression of Gαi3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gβγ-dependent regulation of GIRK by Na+. We propose that Na+ may act as a regulatory factor, or even a second messenger, that regulates effectors via Gβγ.

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K+ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gαi/o. Whether and how Gαi/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gαi3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gαi3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gαi3GDP and, unexpectedly, to Gαi3GTP. i-FRET showed that both Gαi3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a “constitutively active” Gαi3 mutant and Gβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gαi3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between GαiGTP and Gβγ. Our findings imply that Gαi/o and the Gαiβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters.

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits.

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GαiGDP and Gβγ

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi 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 Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ 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 Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A 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 Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal 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 Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.

Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by GalphaiGDP and Gbetagamma.

G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein‐coupled receptors (GPCRs). Although Gαi 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 Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the ‘inactive’ GDP‐bound Gαi3GDP and involves the formation of Gαi3βγ 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 Gαi3, Gαi3G203A, which strongly binds Gβγ, but not by the ‘constitutively active’ mutant, Gαi3Q204L, or by Gβγ‐scavenging proteins. Furthermore, regulation by Gαi3G203A 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 Gαi3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a ‘classical’ Gβγ effector, showing low Ibasal and strong Gβγ‐dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ‐induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3GDP reduced Ibasal 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 Gαi3GDP and Gβγ, while GIRK2 is a Gβγ‐effector insensitive to Gαi3GDP.

Regulation of cardiac L-type Ca2+ channel by coexpression of Gαs in Xenopus oocytes

Activation of Gαs via β‐adrenergic receptors enhances the activity of cardiac voltage‐dependent Ca2+ channels of the L‐type, mainly via protein kinase A (PKA)‐dependent phosphorylation. Contribution of a PKA‐independent effect of Gαs has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous Gαs reduced, whereas coexpression of Gαs enhanced, currents via expressed cardiac L‐type channels, independently of the presence of the auxiliary subunits α2/δ or β2A. Coexpression of Gαs did not increase the amount of α1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed β2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP‐dependent decrease was observed. We conclude that coexpression of Gαs, but not its acute activation via β‐adrenergic receptors, enhances the activity of the cardiac L‐type Ca2+ channel via a PKA‐independent effect on the α1C subunit.