Joanne L. Leaney

The G Protein α Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K+ Channels

In neuronal and atrial tissue, G protein-gated inwardly rectifying K+ channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gβγ dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the Gi/o family but not receptors coupled to the stimulatory G protein Gs. We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein α subunits and chimeras between Gi1 and Gs indicate a central role for the G protein α subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K+ channels.

Regulation of a G protein‐gated inwardly rectifying K+ channel by a Ca2+‐independent protein kinase C

1 Members of the Kir3.0 family of inwardly rectifying K+ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by Gβγ subunits released in response to Gi/o‐coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the Gq/11‐coupled M1 and M3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal‐type channels stably expressed in the human embryonic kidney cell line HEK293. 2 We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M1 and M3 receptors. This contrasts with the simple stimulation seen through activation of M2 and M4 receptors. 3 Channel stimulation via M1 but not M3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M1 and M3 receptors was insensitive. In contrast over‐expression of the C‐terminus of phospholipase Cβ1 or a Gq/11‐specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4 The inhibitory effects of M1 and M3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor. 5 The receptor‐mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0‐669 nm). 6 The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε. 7 We also demonstrate that activation of such a pathway via both receptor‐mediated and receptor‐independent means profoundly attenuated subsequent channel stimulation by Gi/o‐coupled receptors. 8 Our data support a role for a Ca2+‐independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M1 and M3 muscarinic receptor stimulation.

THE MOLECULAR ASSEMBLY OF ATP-SENSITIVE POTASSIUM CHANNELS : DETERMINANTS ON THE PORE FORMING SUBUNIT

ATP-sensitive potassium channels form a link between membrane excitability and cellular metabolism. These channels are important in physiological processes such as insulin release and they are an important site of drug action. They are an octomeric complex comprised of four sulfonylurea receptors, a member of the ATP-binding cassette family of proteins, and four Kir 6.0 subunits from the inward rectifier family of potassium channels. We have investigated the nature of the interaction between SUR1 and Kir 6.2 and the domains on the channel responsible for the biochemical and functional manifestations of coupling. The results point to the proximal C terminus determining biochemical interaction in a region that also largely governs homotypic and heterotypic interaction between different Kir family members. While this domain may be necessary for functional communication between the two proteins, it is not sufficient since relative modifications of either the N or C terminus are able to disrupt many aspects of functional coupling mediated by the sulfonylurea receptor.

Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K+ channels

G protein-gated inwardly rectifying K+ (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G protein-coupled receptors (GPCRs) via the inhibitory family of G protein, Gi/o, in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K+ channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein α subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein α subunit. With some combinations of agonist/GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein α subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs.

A Novel Strategy to Engineer Functional Fluorescent Inhibitory G-protein α Subunits

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein α subunits. We have designed Gi/oα subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K+ channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from Gi/o α subunits (Gi1α, Gi2α, Gi3α, and GoAα) and a mutation introduced at Cys−4 rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed Gi/oα protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant Gi/o α subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gβγ dimer by the mutant Gα subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged Gi3α and GoAα α subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.

Differential Phosphoinositide Binding to Components of the G Protein-Gated K+ Channel

The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K+ channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.