Ca(2+) elevation evoked by membrane depolarization regulates G protein cycle via RGS proteins in the heart.

Regulators of G protein signaling (RGS), which act as GTPase activators, are a family of cytosolic proteins emerging rapidly as an important means of controlling G protein-mediated cell signals. The importance of RGS action has been verified in vitro for various kinds of cell function. Their in situ modes of action in intact cells are, however, poorly understood. Here we show that an increase in intracellular Ca(2+) evoked by membrane depolarization controls the RGS action on G protein activation of muscarinic K(+) (K(G)) channel in the heart. Acetylcholine-induced K(G) current exhibits a slow time-dependent increase during hyperpolarizing voltage steps, referred to as "relaxation." This reflects the relief from the decrease in available K(G) channel number induced by cell depolarization. This phenomenon is abolished when an increase in intracellular Ca(2+) is prevented. It is also abolished when a calmodulin inhibitor or a mutant RGS4 is applied that can bind to calmodulin but that does not accelerate GTPase activity. Therefore, an increase in intracellular Ca(2+) and the resultant formation of Ca(2+)/calmodulin facilitate GTPase activity of RGS and thus decrease the available channel number on depolarization. These results indicate a novel and probably general pathway that Ca(2+)-dependent signaling regulates the G protein cycle via RGS proteins.

An inwardly rectifying K(+) channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain.

Glial cells express inwardly rectifying K(+) (Kir) channels, which play a critical role in the buffering of extracellular K(+). Kir4.1 is the only Kir channel so far shown to be expressed in brain glial cells. We examined the distribution of Kir4.1 in rat brain with a specific antibody. The Kir4.1 immunostaining distributed broadly but not diffusely in the brain. It was strong in some regions such as the glomerular layer of the olfactory bulb, the Bergmann glia in the cerebellum, the ependyma, and pia mater, while little activity was detected in white matter of the corpus callosum or cerebellar peduncle. In the olfactory bulb, Kir4.1 immunoreactivity was detected in a scattered manner in about one-half of the glial fibrillary acidic protein-positive astrocytes. Immunoelectron microscopic examination revealed that Kir4.1 channels were enriched on the processes of astrocytes wrapping synapses and blood vessels. These data suggest that Kir4.1 is expressed in a limited population of brain astrocytes and may play a specific role in the glial K(+)-buffering action.

Interaction between the RGS domain of RGS4 with G protein alpha subunits mediates the voltage-dependent relaxation of the G protein-gated potassium channel.

1. In native cardiac myocytes, there is a time dependence to the G protein-gated inwardly rectifying K(+) (K(G)) channel current during voltage steps that accelerates as the concentration of acetylcholine is increased. This phenomenon has been called 'relaxation' and is not reproduced in the reconstituted Kir3.1/Kir3.4 channel in Xenopus oocytes. We have shown that RGS4, a regulator of G protein signalling, restores relaxation to the reconstituted Kir3.1/Kir3.4 channel. In this study, we examined the mechanism of this phenomenon by expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and mutants of RGS4 in Xenopus oocytes. 2. RGS4 restored relaxation to K(G) channels activated by the pertussis toxin (PTX)-sensitive G protein-coupled m(2)-muscarinic receptor but not to those activated by the G(s) protein-coupled beta(2)-adrenergic receptor. 3. RGS4 induced relaxation not only in heteromeric K(G) channels composed of Kir3.1 and Kir3.4 but also in homomeric assemblies of either an active mutant of Kir3.1 (Kir3.1/F137S) or an isoform of Kir3.2 (Kir3.2d). 4. Truncation mutants of RGS4 showed that the RGS domain itself was essential to reproduce the effect of wild-type RGS4 on the K(G) channel. 5. The mutation of residues in the RGS domain which interact with the alpha subunit of the G protein (G(alpha)) impaired the effect of RGS4. 6. This study therefore shows that interaction between the RGS domain and PTX-sensitive G(alpha) subunits mediates the effect of RGS4 on the agonist concentration-dependent relaxation of K(G) channels.

Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K(+) current (I(K(ACh))). Evidence for functional homomeric GIRK4 channels.

K(+) channels composed of G-protein-coupled inwardly rectifying K(+) channel (GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and various endocrine tissues. They are involved in inhibiting excitability and contribute to regulating important physiological functions such as cardiac frequency and secretion of hormones. The functional cardiac (K((ACh))) channel activated by G(i)/G(o)-coupled receptors such as muscarinic M(2) or purinergic A(1) receptors is supposed to be composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2) fashion. In the present study, we have manipulated the subunit composition of the K((ACh)) channels in cultured atrial myocytes from hearts of adult rats by transient transfection of vectors encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs and investigated the effects on properties of macroscopic I(K(ACh)). Transfection with a GIRK1 vector did not cause any measurable effect on properties of I(K(ACh)), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of activation. Transfection of myocytes with a construct encoding for a concatemeric GIRK4(2) subunit had similar effects on desensitization and inward rectification. Following transfection of a tetrameric construct (GIRK4(4)), these changes in properties of I(K(ACh)) were still observed but were less pronounced. Heterologous expression in Chinese hamster ovary cells and human embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4 resulted in robust currents activated by co-expressed A(1) and M(2) receptors, respectively. These data provide strong evidence that homomeric GIRK4 complexes form functional G(beta)gamma gated ion channels and that kinetic properties of GIRK channels, such as activation rate, desensitization, and inward rectification, depend on subunit composition.

Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium.

1. The inwardly rectifying K+ channel current (IK(IR)) recorded from isolated retinal pigmented epithelial (RPE) cells showed poor dependence on external K+ ([K+]o) and low sensitivity to block by Ba2+. We examined the molecular identity and specific subcellular localization of the KIR channel in RPE cells. 2. The Kir7.1 channel current heterologously expressed in HEK293T cells (human embryonic kidney cell line) showed identical properties to those of the RPE IK(IR), i.e. poor dependence on [K+]o and low sensitivity to Ba2+ block. 3. Expression of Kir7.1 mRNA and protein was detected in RPE cells by RT-PCR and immunoblot techniques, respectively. 4. Immunohistochemical studies including electron microscopy revealed that the Kir7.1 channel was localized specifically at the proximal roots of the apical processes of RPE cells, where Na+,K+-ATPase immunoreactivity was also detected. 5. The middle-distal portions of apical processes of RPE cells in the intact tissue exhibited immunoreactivity of Kir4.1, a common KIR channel. In the isolated RPE cells, however, Kir4.1 immunoreactivity was largely lost, while Kir7.1 immunoreactivity remained. 6. These data indicate that the only IK(IR) recorded in isolated RPE cells is derived from the functional Kir7.1 channel localized at the root of apical processes. Co-localization with Na+,K+-ATPase suggests that the Kir7.1 channel may provide the pathway for recycling of K+ to maintain pump activity and thus is essential for K+ handling in RPE cells.

C-terminal tails of sulfonylurea receptors control ADP-induced activation and diazoxide modulation of ATP-sensitive K(+) channels.

The ATP-sensitive K(+) (K(ATP)) channels are composed of the pore-forming K(+) channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle-type K(ATP) channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K(ATP) channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K(ATP) channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K(ATP) channels containing different SURs.

A regulator of G protein signalling (RGS) protein confers agonist-dependent relaxation gating to a G protein-gated K+ channel.

1. The effects of RGS4 on the voltage-dependent relaxation of G protein-gated K+ (KG) channels were examined by heterologous expression in Xenopus oocytes. 2. While the relaxation kinetics was unaffected by the acetylcholine concentration ([ACh]) in the absence of RGS4, it became dependent on [ACh] when RGS4 was co-expressed. 3. Kinetic analyses indicated that RGS4 confers to the KG channel a voltage-independent inhibitory gating mechanism, which was attenuated by ACh in a concentration-dependent fashion. 4. In vitro biochemical studies showed that RGS4 could bind to the protein complex containing KG channel subunits. 5. Since the native cardiac KG channel exhibited similar agonist-dependent relaxation kinetics to that mediated by RGS4, it is suggested that KG channel gating is a novel physiological target of RGS protein-mediated regulation.

In vivo formation of a proton-sensitive K+ channel by heteromeric subunit assembly of Kir5.1 with Kir4.1.

Kir5.1 is an inwardly rectifying K+ channel (Kir) subunit, whose physiological function is unknown. Human embryonic kidney HEK293T cells co-transfected with rat Kir5.1 and Kir4.1 cDNA expressed a functional K+ channel, whose properties were significantly different from those of the homomeric Kir4.1 channel. Formation of a Kir4. 1/Kir5.1 assembly in HEK293T was confirmed biochemically. We found that heteromeric Kir4.1/Kir5.1 channel activity was affected by internal pH levels between 6.0 and 8.0, when the homomeric Kir4.1 channel activity was relatively stable. Changing external pH in this range had no effect on either Kir channel. Western blot analysis using specific antibodies revealed that Kir4.1 and Kir5.1 proteins were expressed in kidney and brain, but co-immunoprecipitated only from kidney. These results indicate that the co-assembly of Kir5.1 with Kir4.1 occurs in vivo, at least in kidney. The heteromeric Kir4. 1/Kir5.1 channel may therefore sense intracellular pH in renal epithelium and be involved in the regulation of acid-base homeostasis.

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.

Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis.

1. One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G-protein-gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2. Testicular membrane contained a protein that was recognized by the antibody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was approximately 45 kDa, which was smaller than that of Kir3.2c ( approximately 48 kDa). The immunoprecipitant obtained from testis with aG2C-3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N-terminus common to the known Kir3.2 isoforms (aG2N-2). 3. A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be approximately 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N-2. 4. In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G-protein-gated inwardly rectifying K+ channel activity. 5. Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3. 2d, may assemble to form a homomultimeric G-protein-gated K+ channel and be involved in the development of the acrosome during spermiogenesis.

Expression and polarized distribution of an inwardly rectifying K+ channel, Kir4.1, in rat retinal pigment epithelium.

1. In the eye, different substances and ions including potassium (K+) are transported between neural retina and choroid via the subretinal space. Inwardly rectifying K+ channels (Kir) on the apical membrane of retinal pigment epithelial (RPE) cells are thought to play an essential role in K+ transport in the subretinal space. 2. Single-channel recordings from the apical membrane of RPE cells exhibited functional expression of a Kir channel with properties identical to those of Kir4.1, while recordings from the basolateral membrane showed no detectable Kir channel currents. 3. The expression of Kir4.1 mRNA in RPE cells was confirmed by RT-PCR analysis and in situ hybridization. Furthermore, using immunohistochemistry, we found that Kir4.1 was prominently expressed in RPE cells and localized specifically on the processes on their apical membrane. 4. Developmental studies revealed that expression of Kir4.1 started to appear 10 days or later after birth in RPE cells, in parallel with the maturation of retinal neuronal activity as represented by the a- and b-waves of the electroretinogram. 5. These data suggest that Kir4.1 is one of the Kir channels involved in RPE-mediated control of K+ ions in the subretinal space.

Expression of an inwardly rectifying K(+) channel, Kir4.1, in satellite cells of rat cochlear ganglia.

Satellite cells are glial cells wrapped around somata of sensory and autonomic ganglion neurons. Neither their functional roles nor electrical properties have been fully clarified so far. Using immunohistochemistry, we found that inwardly rectifying K(+) channel subunit Kir4.1 (also called Kir1.2 or K(AB)-2) was expressed prominently in the satellite cells of cochlear ganglia. The Kir4.1 immunoreactivity was localized specifically at the myelin sheaths of satellite cells wrapping the somata of the ganglion neurons. Developmental expression of Kir4.1 in satellite cells paralleled development of the action potential in the auditory nerve. These results suggest that this channel in satellite cells may be responsible for the regulation of K(+) extruded from the ganglion neurons during excitation.

Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.

Postembedding immunogold labeling was used to examine the subcellular distribution of the inwardly rectifying K+ channel Kir4.1 in rat retinal Müller cells and to compare this with the distribution of the water channel aquaporin-4 (AQP4). The quantitative analysis suggested that both molecules are enriched in those plasma membrane domains that face the vitreous body and blood vessels. In addition, Kir4. 1, but not AQP4, was concentrated in the basal approximately 300-400 nm of the Müller cell microvilli. These data indicate that AQP4 may mediate the water flux known to be associated with K+ siphoning in the retina. By its highly differentiated distribution of AQP4, the Müller cell may be able to direct the water flux to select extracellular compartments while protecting others (the subretinal space) from inappropriate volume changes. The identification of specialized membrane domains with high Kir4.1 expression provides a morphological correlate for the heterogeneous K+ conductance along the Müller cell surface.

Somatostatin induces hyperpolarization in pancreatic islet alpha cells by activating a G protein-gated K+ channel.

Somatostatin inhibits glucagon-secretion from pancreatic alpha cells but its underlying mechanism is unknown. In mouse alpha cells, we found that somatostatin induced prominent hyperpolarization by activating a K+ channel, which was unaffected by tolbutamide but prevented by pre-treating the cells with pertussis toxin. The K+ channel was activated by intracellular GTP (with somatostatin), GTPgammaS or Gbetagamma subunits. It was thus identified as a G protein-gated K+ (K(G)) channel. RT-PCR and immunohistochemical analyses suggested the K(G) channel to be composed of Kir3.2c and Kir3.4. This study identified a novel ionic mechanism involved in somatostatin-inhibition of glucagon-secretion from pancreatic alpha cells.

Secretagogue-induced exocytosis recruits G protein-gated K+ channels to plasma membrane in endocrine cells.

Stimulation-regulated fusion of vesicles to the plasma membrane is an essential step for hormone secretion but may also serve for the recruitment of functional proteins to the plasma membrane. While studying the distribution of G protein-gated K+ (KG) channels in the anterior pituitary lobe, we found KG channel subunits Kir3.1 and Kir3.4 localized on the membranes of intracellular dense core vesicles that contained thyrotropin. Stimulation of these thyrotroph cells with thyrotropin-releasing hormone provoked fusion of vesicles to the plasma membrane, increased expression of Kir3.1 and Kir3.4 subunits in the plasma membrane, and markedly enhanced KG currents stimulated by dopamine and somatostatin. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for hormone secretion in the endocrine system.

Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra.

G-protein-gated K+ (KG) channels generate slow inhibitory postsynaptic potentials in the brain. Current opinion suggests that neuronal KG channels are heterotetramers of Kir3.1 and Kir3.2. In substantia nigra (SN), however, mRNA of Kir3.1 does not express, whereas that of Kir3.2 clearly does. Therefore, we have characterized the KG channels containing Kir3.2 subunits in SN using biochemical and immunological techniques. We found that they were composed of only Kir3.2 subunits and did not contain significant amounts of either Kir3.1 or Kir3.3. Furthermore, at least some of the KG channels in SN were assemblies of the splicing variants Kir3. 2a and Kir3.2c. The channels were localized specifically at the postsynaptic membrane on the dendrites of dopaminergic neurons. Kir3. 2c, but not Kir3.2a, could bind a PDZ domain-containing protein, PSD-95. The heterologously expressed KG channels composed of Kir3.2a plus Kir3.2c or Kir3.2a alone were activated by G-protein stimulation, but expression of Kir3.2c alone was not. This study reveals that the Kir3.2 splicing variants play distinct roles in the control of function and localization of some of the KG channels in dopaminergic neurons of SN.

A novel junction-like membrane complex in the optic nerve astrocyte of the Japanese macaque with a possible relation to a potassium ion channel.

BACKGROUND: A new type of junction-like membrane complex (JMC) was detected between adjacent astrocytes in the optic nerve of Japanese macaque (macaca fuscata). This membrane complex morphologically resembled a cell junction, but a possible role for potassium ion channels could not be denied based on freeze-fracture replica observation. We attempted to determine the chemical nature and function of the novel JMC. METHODS: Using an electron microscope, we observed JMCs in the optic nerve astrocyte. In addition, we observed them using a freeze-fracture replica and immunohistochemistry with connexin 43, a gap junction specific protein. Furthermore, immunolocalization of an inwardly rectifying potassium ion channel, K(AB)-2 (Kir4.1), was studied with a confocal laser-scanning microscope, and an electron microscope using a newly developed pre-embedding method. RESULTS: These JMCs were abundant around the blood vessel in the area just behind the lamina cribrosa. At JMCs the inner leaflet was thicker than the outer leaflet and electron-dense materials were packed in the intercellular space. Freeze-fracture replica observation revealed orthogonal arrays of particles, probably at the place of JMCs, that have been considered a potassium ion channel. No connexin 43 immunoreactivity was detected in JMCs, while K(AB)-2 was mostly localized on either side of the opposing cell membranes of JMC. CONCLUSIONS: These JMCs do not seem to be a simple junction, but relate to a potassium ion channel. The area just behind the lamina cribrosa may be important in terms of conductance of the optic nerve impulse.

Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Müller cell membrane: their regulation by insulin and laminin signals.

Inwardly rectifying potassium (K+) channels (Kir) in Müller cells, the dominant glial cells in the retina, are supposed to be responsible for the spatial buffering action of K+ ions. The molecular properties and subcellular localization of Müller cell Kir channels in rat and rabbit retinas were examined by using electrophysiological, molecular biological, and immunostaining techniques. Only a single population of Kir channel activity, the properties of which were identical to those of KAB-2/Kir4.1 expressed in HEK293T cells, could be recorded from endfoot to the distal portion of Müller cells. Consistently, Northern blot, in situ hybridization, and RT-PCR analyses indicated expression of Kir4. 1 in Müller cells per se. The Kir4.1 immunoreactivity was distributed in clusters throughout Müller cell membrane. The Kir4.1 expression in Müller cells disappeared promptly after culturing. When the dissociated Müller cells were cultured on laminin-coated dishes in the presence of insulin, Kir4.1 immunoreactivity was detected in a clustered manner on the cell membrane. Because insulin and laminin exist in the surrounding of Müller cells in the retina, these substances possibly may be physiological regulators of expression and distribution of Kir4.1 in Müller cells in vivo.

An ATP-dependent inwardly rectifying potassium channel, KAB-2 (Kir4. 1), in cochlear stria vascularis of inner ear: its specific subcellular localization and correlation with the formation of endocochlear potential.

Cochlear endolymph has a highly positive potential of approximately +80 mV. This so-called endocochlear potential (EP) is essential for hearing. Although pivotal roles of K+ channels in the formation of EP have been suggested, the types and distribution of K+ channels in cochlea have not been characterized. Because EP was depressed by vascular perfusion of Ba2+, an inhibitor of inwardly rectifying K+ (Kir) channels, but not by either 4-aminopyridine or tetraethylammonium, we examined the expression of Kir channel subunits in cochlear stria vascularis, the tissue that is supposed to play the central role in the generation of positive EP. Of 11 members of the Kir channel family examined with reverse transcription-PCR, we could detect only expression of KAB-2 (Kir4.1) mRNA in stria vascularis. KAB-2 immunoreactivity was specifically localized at the basolateral membrane of marginal cells but not in either basal or intermediate cells. Developmental expression of KAB-2 in marginal cells paralleled formation of EP. Furthermore, deaf mutant mice (viable dominant spotting; WV/WV) expressed no KAB-2 in their marginal cells. These results suggest that KAB-2 in marginal cells may be critically involved in the generation of positive EP.