Rapid desensitization of G protein-gated inwardly rectifying K(+) currents is determined by G protein cycle

Activation of G protein-gated inwardly rectifying K⁺ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of G_i/o-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several G_i/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A₁, adrenergic _2A, dopamine D_2S, M₄ muscarinic, and GABA_B1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A₁ receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca²⁺ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific G subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle. G protein-coupled receptor; potassium channel; inward rectifier; kinetics     

Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

Critical determinants of the G protein gamma subunits in the Gbetagamma stimulation of G protein-activated inwardly rectifying potassium (GIRK) channel activity

The betagamma subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions. Although GIRK currents are stimulated by mammalian Gbetagamma subunits, we show that they were inhibited by the yeast Gbetagamma (Ste4/Ste18) subunits. A chimera between the yeast and the mammalian Gbeta1 subunits (ymbeta) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast Ggamma subunits, respectively. This result underscores the critical functional influence of the Ggamma subunits on the effectiveness of the Gbetagamma complex. A series of chimeras between Ggamma2 and the yeast Ggamma revealed that the C-terminal half of the Ggamma2 subunit is required for channel activation by the Gbetagamma complex. Point mutations of Ggamma2 to the corresponding yeast Ggamma residues identified several amino acids that reduced significantly the ability of Gbetagamma to stimulate channel activity, an effect that was not due to improper association with Gbeta. Most of the identified critical Ggamma residues clustered together, forming an intricate network of interactions with the Gbeta subunit, defining an interaction surface of the Gbetagamma complex with GIRK channels. These results show for the first time a functional role for Ggamma in the effector role of Gbetagamma.     

Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels

Activation of the heteromeric G protein-gated inwardly rectifying K(+) channel (GIRK) GIRK1 and GIRK4 subunits gives rise to I(KACh), which controls excitability in atrial tissue. Although homomeric GIRK4 channels localize to the plasma membrane and display moderate function, GIRK1 channels fail to localize to the cell surface and do not exhibit significant function as homomers. Using oocytes to express GFP-tagged GIRK1 and GIRK4 and chimeras between these two proteins, we have identified two regions, one in the proximal C terminus and another in the distal N terminus that are critical for their subcellular localization. Replacement of both of these regions in GIRK1 with corresponding regions from GIRK4 was required for efficient expression of GIRK1 on the plasma membrane. Replacement of either region by itself was ineffective. The distal N terminus and proximal C terminus have been previously suggested to play important roles in ER-export and subunit co-assembly respectively in this family of channels. Our data indicate for the first time that both of these regions need to work in concert to mediate efficient targeting of these channels to the plasma membrane.     

The (beta)gamma subunits of G proteins gate a K(+) channel by pivoted bending of a transmembrane segment

The molecular mechanism of ion channel gating remains unclear. Using approaches such as proline scanning mutagenesis and homology modeling, we localize the gate of the K(+) channels controlled by the (beta)gamma subunits of G proteins at the pore-lining bundle crossing of the second transmembrane (TM2) helices. We show that the flexibility afforded by a highly conserved glycine residue in the middle of TM2 is crucial for channel gating. In contrast, flexibility introduced immediately below the gate disrupts gating. We propose that the force produced by channel-G(beta)gamma interactions is transduced through the rigid region below the helix bundle crossing to bend TM2 at the glycine that serves as a hinge and open the gate.     

Regulation of the inward rectifying properties of G-protein-activated inwardly rectifying K+ (GIRK) channels by Gbeta gamma subunits

Gbetagamma subunits are known to bind to and activate G-protein-activated inwardly rectifying K(+) channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (I(KACh)) channels in feline atrial myocytes or heterologously expressed GIRK1/4 channels in Chinese hamster ovary cells and HEK 293 cells uncovered a novel Gbetagamma subunit mediated regulation of the inwardly rectifying properties of these channels. I(KACh) activated by submaximal concentrations of acetylcholine exhibited a approximately 2.5-fold stronger inward rectification than I(KACh) activated by saturating concentrations of acetylcholine. Similarly, the inward rectification of currents through GIRK1/4 channels expressed in HEK cells was substantially weakened upon maximal stimulation with co-expressed Gbetagamma subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when Gbetagamma was over-expressed. The Gbetagamma induced weakening of inward rectification was associated with reduced potencies for Ba(2+) and Cs(+) to block channels from the extracellular side. Based on these results we propose that saturation of the channel with Gbetagamma leads to a conformational change within the pore of the channel that reduced the potency of extracellular cations to block the pore and increased the fraction of channels inert to a pore block in outward direction.     

The G protein alpha 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 Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). 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 alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.     

Cytoplasmic unsaturated free fatty acids inhibit ATP-dependent gating of the G protein-gated K(+) channel

This study reports the identification of an endogenous inhibitor of the G protein-gated (K(ACh)) channel and its effect on the K(ACh) channel kinetics. In the presence of acetylcholine in the pipette, K(ACh) channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated K(ACh) channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the K(ACh) channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of approximately 40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.2-2 microM) reversibly inhibited the ATP-dependent gating of native K(ACh) channels in atrial cells and hippocampal neurons, and of recombinant K(ACh) channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4, 5-bisphosphate (PIP(2))-induced changes in K(ACh) channel kinetics but were ineffective against ATP-activated background K(1) channels and PIP(2)-activated K(ATP) channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal K(ACh) channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G protein-gated K(+) channel.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes

The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.     

Anchoring proteins confer G protein sensitivity to an inward-rectifier K(+) channel through the GK domain

Anchoring proteins cluster receptors and ion channels at postsynaptic membranes in the brain. They also act as scaffolds for intracellular signaling molecules including synGAP and NO synthase. Here we report a new function for intracellular anchoring proteins: the regulation of synaptic ion channel function. A neuronal G protein-gated inwardly rectifying K(+) channel, Kir3.2c, can not be activated either by M(2)-muscarinic receptor stimulation or by G(betagamma) overexpression. When coexpressed with SAP97, a member of the PSD/SAP anchoring protein family, the channel became sensitive to G protein stimulation. Although the C-terminus of Kir3. 2c bound to the second PDZ domain of SAP97, functional analyses revealed that the guanylate kinase (GK) domain of SAP97 is crucial for sensitization of the Kir3.2c channel to G protein stimulation. Furthermore, SAPAP1/GKAP, which binds specifically to the GK domain of membrane-associated guanylate kinases, prevented the SAP97-induced sensitization. The function of a synaptic ion channel can therefore be controlled by a network of various intracellular proteins.     

Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits

Novel G protein alpha subunits were purified from rat brain by an affinity matrix containing immobilized beta gamma subunits (Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These alpha subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known alpha subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other alpha subunits. Sequences are identical with one or more members of a new family of alpha subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an alpha subunit labeled alpha q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP gamma S only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new alpha subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.     

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.     

Calcium-sensing receptor decreases cell surface expression of the inwardly rectifying K+ channel Kir4.1

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.