GIRK channels: hierarchy of control. Focus on "PKC-{delta} sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells"

Gerda E. Breitwieser

Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania

G PROTEIN-ACTIVATED inwardly rectifying K+ channels (GIRK channels) contribute to regulation of membrane excitability in muscle and nerve. There are four distinct mammalian genes in this subfamily of inward rectifier K+ channels (termed GIRK1-GIRK4 or Kir3.1-Kir3.4, 60–80% identity between family members), and functional channels are comprised of a dimer of heterodimers containing Kir3.1 plus any of the other subunits Kir3.2-Kir3.4 (reviewed in Ref. 4). Before their molecular cloning, the core signaling pathway, which regulates GIRK channels in many tissues was distilled to a three component system: a G protein-coupled receptor (GPCR), a pertussis toxin-sensitive heterotrimeric G protein (Gi) releasing G{beta}{gamma}-subunits, and a G{beta}{gamma}-gated ion channel (4). Since the molecular identification of GIRK channels, however, the list of regulators of GIRK channel activation has grown to include ATP (plus Mg2+), Na+, and phosphatidylinositol 4,5-bisphosphate (PIP2) (4). The current focus article (Ref. 1; See p. C543 in this issue) addresses two of these regulators, ATP + Mg2+ (supporting phosphorylation) and PIP2.

Cross-talk between Gi-coupled GPCRs which activate GIRK channels and Gq-coupled GPCRs, which can inhibit GIRK channels, has been observed both in vivo and when channel subunits are expressed in mammalian cell lines (reviewed in Ref. 8). The molecular mechanism of Gq-mediated inhibition includes, at a minimum, G{alpha}q-subunits, phospholipase C (PLC), which causes decreases in the PIP2 concentration, and PKC, but does not require the more distal messengers inositol 1,4,5-trisphosphate or Ca2+. Studies of Gq-linked inhibition have reached divergent conclusions, with significant support being marshaled for regulation of GIRK channels by either PKC-mediated phosphorylation (1, 7, 911) or by PLC-mediated PIP2 depletion (1–3, 5, 8). The results by Brown et al. (1) in the current article in focus bring together these divergent mechanisms, and suggest a hierarchy of regulation of GIRK channels by two consequences of Gq activation, namely, PKC activation and dynamic changes in PIP2 levels.

Gq-linked GPCRs induce activation of PKC, which phosphorylates GIRK1 both in vitro and in vivo (1, 10). Functional studies (1, 7, 911) have demonstrated inhibition of GIRK channels after PKC activation by phorbol esters or diacylglycerol and resistance to Gq-mediated inhibition in the presence of PKC inhibitors. These strategies for modulation of PKC activity are, however, not uniformly effective in altering GIRK channel inhibition, leading some authors to the conclusion that PKC phosphorylation of GIRK channels is not involved in Gq-mediated inhibition (2, 3, 5, 8). Signaling in isolated cardiac myocytes is dependent on physiological levels of channels and PKC isoforms, whereas transfected mammalian cells can have a large excess of GIRK channels with potentially altered regulation due to limiting levels of required scaffold proteins. This simplistic explanation does not suffice, however, because PKC-mediated effects have been observed in both isolated cardiac myocytes (11) and in Xenopus oocytes (9). It is more likely that the protocol used to test for the effects of PKC is critical, particularly if GIRK channel phosphorylation is constitutive and/or permissive for other more dynamic regulatory interactions, such as those with G{beta}{gamma} and/or PIP2.

Gq pathway stimulation also results in depletion of membrane PIP2, the substrate for PI-PLC. PIP2 has emerged, in the past decade, as a modulator of the function of many ion channels and transporters (for review, see Ref. 5). GIRK channels require interaction with PIP2 for activity, and depletion by PLC activation inhibits the channel (2, 6). The carboxyl terminus of GIRK channels binds both PIP2 and G{beta}{gamma}, stabilizing the activated state (6). PLC-mediated depletion of PIP2 inhibits GIRK channels both in atrial myocytes and transfected mammalian cells (2, 3, 5, 8). Divergent efficacies of a variety of Gq-coupled receptors in mediating GIRK channel inhibition via PIP2 depletion may result from differences in subcellular localizations of receptors relative to PLC and GIRK channels (2). PIP2 has been termed a silent partner in protein activation because its levels might not change sufficiently during normal receptor activation to perturb binding (5). Indeed, isolated cardiac myocytes contain significantly less PIP2 than intact cardiac tissue, in part as a result of isolation-dependent loss of cell contact- and/or matrix-dependent signaling pathways, which stimulate lipid kinases. Prolonged vagal stimulation of intact atria may not significantly decrease bulk PIP2 levels (Ref. 5 and references therein). Whereas bulk levels of PIP2 may be stable, it is likely that PIP2 levels are dynamically modulated in the vicinity of PLC. Resolution of local changes in PIP2 in the vicinity of GIRK channels will be required to fully test for receptor-specific PIP2 regulation, perhaps utilizing fluorescence or bioluminescence resonance energy transfer technology with labeled PIP2 and channel subunits.

The current article in focus by Brown et al. (1) resolves the differences between the PKC and PIP2 models for GIRK channel inhibition and establishes a hierarchy of regulation of GIRK channel activation, demonstrating that PKC phosphorylation of GIRK1 (Kir3.1) subunits modulates channel sensitivity to PIP2. Using human embryonic kidney (HEK)-293 cells stably transfected with Kir3.1/3.2 channels, they demonstrate PKC mediates the inhibition of GIRK1/2 channels, whereas PIP2 is required for recovery from inhibition. Critical to these studies is the use of the perforated patch recording mode, which prevents loss of PIP2 metabolites to the pipette. In whole cell recordings, PIP2 in the pipette solution attenuated the inhibitory effect of Gq activation. PKC inhibitors are rather blunt tools with limited isoform specificity. To determine which PKCs inhibit GIRK1/2 channels, the authors (1, 7) generated expression constructs for all of the isoforms they previously demonstrated are expressed in HEK-293 cells. PKC-{beta}I and PKC-{delta} had inhibitory effects on GIRK1/2 channel currents. Interestingly, expression controls demonstrated that PKC-{delta} was the most poorly expressed isoform. To clarify the relative importance of the two isoforms, the authors did two critical and complementary experiments. First, they equalized the expression levels of the two isoforms (requiring a radical reduction in PKC-{beta}I levels); under these conditions, only PKC-{delta} was able to modulate GIRK activity. Second, they demonstrated that PKC-{delta} was translocated to the membrane on M3 receptor activation. Translation of these results to cardiac myocytes or neurons would require determination of the relative endogenous expression levels of PKC-{beta}I and PKC-{delta}; the present article in focus narrows the field of isoforms that must be examined.

If PKC-{delta} is involved in GIRK channel inhibition, is the channel itself phosphorylated? In excised patches, exogenous purified PKC (in the presence of ATP + Mg2+) inhibited GIRK currents; recovery was prevented by okadaic acid, a protein phosphatase inhibitor. GIRK1/2 gating by G{beta}{gamma} subunits required prior phosphorylation by PKC, suggesting phosphorylation is a prerequisite for G{beta}{gamma}-mediated gating. While these results point to a highly localized site for PKC phosphorylation, they do not restrict the mechanism to phosphorylation of GIRK channels. The authors demonstrate that the carboxyl terminus of Kir3.1 is heavily phosphorylated by PKC while the carboxyl terminus of Kir3.2 is not. As an initial step in identifying critical residues, they generated the mutations in Kir3.1(S185A) and Kir3.2(S178A), which eliminated PMA inhibition of Kir3.1/Kir3.4 channels (9). Unfortunately, Kir3.1(S185A)/Kir3.2(S178A) channels were still inhibited by carbachol, PKC, and PMA. It remains to be determined, therefore, whether other or additional consensus sites for PKC phosphorylation on the Kir3.1/Kir3.2 channels are required for PKC-mediated inhibition, or whether a closely associated regulatory protein is the target of PKC-{delta}.

Overall, the results presented in the article in focus (1) provide a new starting point for understanding GIRK channel regulation by Gq-coupled GPCRs, as summarized in Fig. 1. Gi-coupled GPCRs activate GIRK channels by releasing G{beta}{gamma}-subunits, which interact with and gate the channel in the presence of PIP2 (Fig. 1A). When both Gi-coupled and Gq-coupled GPCRs are activated, initial stimulation proceeds as in Fig. 1A, but G{alpha}q-mediated activation of PLC reduces the concentration of PIP2 in the vicinity of the channel and increases diacylglycerol, recruiting PKC-{delta} to the membrane. Phosphorylation of GIRK channels prevents channel activation by PIP2. One possibility, which must be experimentally tested, is that incorporation of multiple phosphates into Kir3.1 reduces channel affinity for PIP2. Figure 1B illustrates the key interactions schematically. There are, of course, many questions remaining, including the identification of the site(s) for PKC-{delta} phosphorylation on GIRK subunits and the alterations in channel conformation and/or interactions with G{beta}{gamma}-subunits that result. Higher-order questions include the contributions of signaling complexes to the process, as well as the dynamics of PIP2 depletion and recovery in the vicinity of the channel. Whether PKC-{delta}-mediated phosphorylation or PIP2 depletion is the dominant regulator of GIRK activity in vivo remains to be determined, but it is interesting to note that a recent report suggests that PKC-{delta} expression is increased in chronically hypoxic rat myocardium, and may contribute to adaptive cardioprotection (12).



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Fig. 1. Mechanism for Gq-mediated inhibition of G protein activated inwardly rectifying K+ (GIRK) channels. A: activation of Gi protein-coupled receptors (GPCRs) results in release of G{beta}{gamma}-subunits, which activate GIRK channel heteromers in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2). B: simultaneous stimulation of both Gi- and Gq-coupled GPCRs activates GIRK initially, but G{alpha}q stimulation of phospholipase C (PLC) decreases PIP2 and increases diacylglycerol (DAG), which recruits PKC-{delta}. Phosphorylation of GIRK subunits results in decreased interaction with PIP2 and inhibition. Recovery after agonist removal (not shown) requires resynthesis of PIP2, and dephosphorylation of the channel. IP3, inositol 1,4,5-trisphosphate.

 


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. E. Breitwieser, Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822 (e-mail: gebreitwieser{at}geisinger.edu)


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