Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism.
(41/2415)
ROMK inward-rectifier K+ channels control renal K+ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4, 5-bisphosphate (PIP2) is essential for channel activation, we investigate here the role of PIP2 in regulation of ROMK1 by PKA. By using adenosine-5'-[gamma-thio]triphosphate) (ATP[gammaS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP2. Rather, phosphorylation by PKA + ATP[gammaS] lowers the concentration of PIP2 necessary for activation of the channels. In solution-binding assays, anti-PIP2 antibodies bind PIP2 and prevent PIP2-channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP2 and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP2 interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP2-channel interaction. (+info)
New roles for RGS2, 5 and 8 on the ratio-dependent modulation of recombinant GIRK channels expressed in Xenopus oocytes.
(42/2415)
1. The activation of G protein-regulated inward rectifying potassium (GIRK) channels is modulated by G protein-coupled receptors (GPCRs) via the G protein betagamma subunits and is accelerated by regulators of G protein signalling (RGS). In the present study we investigated the ratio dependence of receptor-mediated activation and deactivation and the influence of new members of the RGS protein family on GIRK currents by coexpressing the recombinant protein subunits in Xenopus oocytes and further analysis of the whole cell currents. 2. The activation of GIRK channels by the muscarinic acetylcholine receptor M2 (M2 mAChR) is strongly dependent on the ratio of receptor to channel in Xenopus oocytes. The increase and on-rate of the amplified current is affected by this ratio. An excess of receptor over channel is necessary for current amplification, while the reverse excess of channel over receptor abolishes the effect. 3. The speed of receptor-mediated activation of GIRK currents is accelerated for a high ratio of receptor to channel, while the time of deactivation is independent of this ratio. 4. Coexpression of RGS2, 5 and 8 accelerates the speed for ACh-mediated activation and deactivation of GIRK1/2 and GIRK1/4 currents. Thereby the receptor/channel/RGS ratio determines the amount of current amplification. 5. Bordetella pertussis toxin completely abolished ACh-mediated current amplification of GIRK channels coexpressed with or without RGS2. 6. Two single point mutations in the RGS2 protein (RGS2(N109S) and RGS2(L180F)) reduced the acceleration of current amplification after ACh application on GIRK1/4 channels compared with RGS2 wild-type protein. (+info)
G protein-activated inwardly rectifying K+ (GIRK) currents in dendrites of rat neocortical pyramidal cells.
(43/2415)
1. We performed patch-clamp recordings on acutely isolated dendritic segments and cell somata of rat neocortical pyramidal neurons to determine and compare the relative density of G protein-activated K+ (GIRK) currents in the two cellular compartments. 2. Hyperpolarizing voltage ramps and elevation of extracellular K+ concentration to 40 mM served to identify inwardly rectifying K+ currents. Near-saturating concentrations of adenosine (100 microM), baclofen (20 microM) and serotonin (20 microM) all produced robust GIRK currents in cell somata as well as in dendritic segments that were completely abolished by Ba2+ (200 microM). In addition to agonist-activated GIRK currents, both somata and dendrites displayed a constitutive Ba2+-sensitive inward rectification. 3. In order to compare the relative strengths of GIRK current responses in the two compartments, GIRK conductance was normalized to surface area. In contrast to intrinsic, G protein-independent inward rectification, which was comparable in size in the two compartments, all three agonists evoked significantly larger GIRK conductances in dendrites than in somata. 4. Our data suggest that several neurotransmitters might employ GIRK currents as a tool to directly modulate the electrical properties of dendrites. In concert with voltage-dependent K+ currents and the hyperpolarization-activated cation current (Ih) of the dendrite, GIRK currents should dampen dendritic excitability and thus influence various aspects of dendritic signal integration. (+info)
A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.
(44/2415)
Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients. (+info)
Localization and age-dependent expression of the inward rectifier K+ channel subunit Kir 5.1 in a mammalian reproductive system.
(45/2415)
Kir 5.1 is a member of the inward rectifier potassium channel superfamily which does not form functional channels when expressed by itself in Xenopus laevis oocytes. rt-PCR reveals high levels of Kir 5.1 mRNA expression in testis but the function of this channel remains unknown. To determine the cell-specific expression of this channel in the testis we raised a polyclonal antibody against an external epitope of Kir 5.1 and tested its specificity in Xenopus oocytes expressing several cloned Kir subunits. Strong immunoreactivity for Kir 5.1 was found in seminiferous tubules of rat testis and, particularly, in spermatogonia, primary and secondary spermatocytes, spermatids and in the head and body of spermatozoa. The intensity of Kir 5.1 immunofluorescence, quantified using laser scanning microscopy, increased with age at every stage in the development of sperm from spermatogonia and reached a peak in 60-day-old rats. In contrast, the immunofluorescence decreased in 90-day-old animals and was detected mostly in spermatozoa. The results demonstrate that Kir 5.1 expression in the testis is localised to cells involved in spermatogenesis, showing a temporal pattern of expression during sexual maturity. (+info)
Identification of the high-affinity tolbutamide site on the SUR1 subunit of the K(ATP) channel.
(46/2415)
ATP-sensitive potassium channels (K(ATP)) are formed from four pore-forming Kir6.2 subunits complexed with four regulatory sulfonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). The sensitivity of the channel to different sulfonylureas depends on the SUR isoform. In particular, Kir6.2-SUR1 but not Kir6.2-SUR2A channels are blocked by tolbutamide with high affinity. We made chimeras between SUR1 and SUR2A to identify the region of the protein involved in high-affinity tolbutamide block. Chimeric SURs were coexpressed with Kir6.2 in Xenopus oocytes, and macroscopic currents were measured in inside-out membrane patches. High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Binding of [3H]glibenclamide to membranes expressing SUR1 was abolished concomitantly with the loss of high-affinity tolbutamide block. These results suggest that a site in the COOH-terminal set of TMs of the SUR1 subunit of the K(ATP) channel is involved in the binding of tolbutamide and glibenclamide. (+info)
Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels.
(47/2415)
A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level. (+info)
Bombesin receptors inhibit G protein-coupled inwardly rectifying K+ channels expressed in Xenopus oocytes through a protein kinase C-dependent pathway.
(48/2415)
Although activation of G protein-coupled inward rectifying K+ (GIRK) channels by Gi/Go-coupled receptors has been shown to be important in postsynaptic inhibition in the central nervous system, there is also evidence to suggest that inhibition of GIRK channels by Gq-coupled receptors is involved in postsynaptic excitation. In the present study we addressed whether the Gq-coupled receptors of the bombesin family can couple to GIRK channels and examined the mechanism by which this process occurs. Different combinations of GIRK channel subunits (Kir3.1, Kir3.2, and Kir3.4) and bombesin receptors (BB1 and BB2) were expressed in Xenopus oocytes. In all combinations tested GIRK currents were reversibly inhibited upon application of the bombesin-related peptides, neuromedin B or gastrin-releasing peptide in a concentration-dependent manner. Incubation of oocytes in the phospholipase C inhibitor U73122 or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine significantly reduced the inhibition of GIRK currents by neuromedin B, whereas the Ca2+ chelator, BAPTA-AM had no effect. The involvement of PKC was further demonstrated by direct inhibition of GIRK currents by the phorbol esters, phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate. In contrast, the inactive phorbol ester 4alpha-phorbol and protein kinase A activators, forskolin and 8-bromo cAMP did not inhibit GIRK currents. At the single-channel level, direct activation of PKC using phorbol ester phorbol-12, 13-dibutyrate caused a dramatic reduction in open probability of GIRK channels due to an increase in duration of the interburst interval. (+info)