ABSTRACT
TWIK-related acid-sensitive K(+) (TASK) channels contribute to setting the resting potential of mammalian neurons and have recently been defined as molecular targets for extracellular protons and volatile anesthetics. We have isolated a novel member of this subfamily, hTASK-5, from a human genomic library and mapped it to chromosomal region 20q12-20q13. hTASK-5 did not functionally express in Xenopus oocytes, whereas chimeric TASK-5/TASK-3 constructs containing the region between M1 and M3 of TASK-3 produced K(+) selective currents. To better correlate TASK subunits with native K(+) currents in neurons the precise cellular distribution of all TASK family members was elucidated in rat brain. A comprehensive in situ hybridization analysis revealed that both TASK-1 and TASK-3 transcripts are most strongly expressed in many neurons likely to be cholinergic, serotonergic, or noradrenergic. In contrast, TASK-5 expression is found in olfactory bulb mitral cells and Purkinje cells, but predominantly associated with the central auditory pathway. Thus, TASK-5 K(+) channels, possibly in conjunction with auxiliary proteins, may play a role in the transmission of temporal information in the auditory system.
Subject(s)
Auditory Pathways/metabolism , Brain/metabolism , Gene Expression Regulation/physiology , Nerve Tissue Proteins , Neurons/metabolism , Potassium Channels, Tandem Pore Domain , Potassium Channels/genetics , Amino Acid Sequence/genetics , Animals , Auditory Pathways/cytology , Base Sequence/genetics , Brain/cytology , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/genetics , Female , Humans , In Situ Hybridization , Membrane Potentials/genetics , Molecular Sequence Data , Neurons/cytology , Oocytes/cytology , Oocytes/metabolism , Potassium Channels/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , XenopusABSTRACT
BACKGROUND: K(+) channels have important functions in the kidney, such as maintenance of the membrane potential, volume regulation, recirculation, and secretion of potassium ions. The aim of this study was to obtain more information on the localization and possible functional role of the inwardly rectifying K(+) channel, Kir7.1. METHODS: Kir7.1 cDNA (1114 bp) was isolated from guinea pig kidney (gpKir7.1), and its tissue distribution was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, a genomic DNA fragment (6153 bp) was isolated from a genomic library. cRNA was expressed in Xenopus laevis oocytes for functional studies. Immunohistochemistry and RT-PCR were used to localize Kir7.1 in guinea pig and human kidney. RESULTS: The expression of gpKir7.1 in Xenopus laevis oocytes revealed inwardly rectifying K(+) currents. The reversal potential was strongly dependent on the extracellular K(+) concentration, shifting from -14 mV at 96 mmol/L K(+) to -90 mV at 1 mmol/L K(+). gpKir7.1 showed a low affinity for Ba(2+). Significant expression of gpKir7.1 was found in brain, kidney, and lung, but not in heart, skeletal muscle, liver, or spleen. Immunocytochemical detection in guinea pig identified the gpKir7.1 protein in the basolateral membrane of epithelial cells of the proximal tubule. RT-PCR analysis identified strong gpKir7.1 expression in the proximal tubule and weak expression in glomeruli and thick ascending limb. In isolated human tubule fragments, RT-PCR showed expression in proximal tubule and thick ascending limb. CONCLUSION: Our results suggest that Kir7.1 may contribute to basolateral K(+) recycling in the proximal tubule and in the thick ascending limb.
Subject(s)
Kidney Tubules, Proximal/chemistry , Loop of Henle/chemistry , Potassium Channels, Inwardly Rectifying , Potassium Channels/analysis , Potassium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cloning, Molecular , Female , Gene Expression/physiology , Guinea Pigs , Humans , Kidney Tubules, Proximal/metabolism , Loop of Henle/metabolism , Male , Membrane Potentials/physiology , Molecular Sequence Data , Oocytes/physiology , Patch-Clamp Techniques , Potassium/metabolism , Potassium Channels/metabolism , RNA, Messenger/analysis , Transfection , Xenopus laevisABSTRACT
ATP-sensitive K (K(ATP)) channels are inhibited by cytosolic ATP, a defining property that implicitly links these channels to cellular metabolism. Here we report a direct link between fatty acid metabolism and K(ATP) channels in cardiac muscle cells. Long-chain (LC) acyl-coenzyme A (CoA) esters are synthesized from fatty acids and serve as the principal metabolic substrates of the heart. We have studied the effects of LC acyl-CoA esters and LC fatty acids on K(ATP) channels of isolated guinea pig ventricular myocytes and compared them with the effects of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Application of oleoyl-CoA (0.2 or 1 micromol/L), a naturally occurring acyl-CoA ester, to the cytosolic side of excised patches completely prevented rundown of K(ATP) channels, but not of Kir2 channels. The open probability of K(ATP) channels measured in the presence of oleoyl-CoA or PIP(2) was voltage dependent, increasing with depolarization. Oleoyl-CoA greatly reduced the ATP sensitivity of K(ATP) channels. At a concentration of 2 micromol/L, oleoyl-CoA increased the half-maximal inhibitory concentration of ATP >200-fold. The time course of the decrease in ATP sensitivity was much faster during application of oleoyl-CoA than during application of PIP(2). The effects of PIP(2), but not of oleoyl-CoA, were inhibited by increasing Ca(2+) to 1 mmol/L. Oleate (C18:1; 10 micromol/L), the precursor of oleoyl-CoA, inhibited K(ATP) channels activated by oleoyl-COA: Palmitoleoyl-CoA and palmitoleate (C16:1) exerted similar reciprocal effects. These findings indicate that LC fatty acids and their CoA-linked derivatives may be key physiological modulators of K(ATP) channel activity in the heart.
Subject(s)
Acyl Coenzyme A/pharmacology , Adenosine Triphosphate/pharmacology , Fatty Acids, Unsaturated/pharmacology , Heart Ventricles/drug effects , Potassium Channels/drug effects , Acyl Coenzyme A/chemistry , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Guinea Pigs , Heart Ventricles/cytology , Kinetics , Membrane Potentials/drug effects , Patch-Clamp Techniques , Phosphatidylinositol 4,5-Diphosphate/pharmacology , Potassium Channels/metabolism , Time Factors , Ventricular FunctionABSTRACT
The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 M for gpKir2.1, 0.51 M for gpKir2.2, 10.26 M for gpKir2.3 and 235 M for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.
Subject(s)
Myocardium/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Animals , Barium/pharmacology , Cell Line , Cloning, Molecular , Electric Conductivity , Guinea Pigs , Humans , Microscopy, Fluorescence , Myocardium/cytology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Potassium Channels/drug effects , Potassium Channels/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Xenopus laevisABSTRACT
BACKGROUND: The renal K(+) channel ROMK (Kir1.1) controls salt reabsorption in the kidney. Loss-of-function mutations in this channel cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), which is characterized by severe renal salt and fluid wasting. METHODS: We investigated 10 HPS/aBS patients for mutations in the ROMK gene by single-strand conformation polymorphism analysis (SSCA) and direct sequencing. To assess the functional consequences, Ba(2+)-sensitive K(+) currents were measured in five mutants of the core region as well as one mutant with truncated C-terminus, using the two-electrode voltage-clamp technique after an injection of mutant cRNA into Xenopus oocytes. RESULTS: Three novel ROMK mutations were identified together with six mutations described previously. The mutations were categorized into three groups: (1) amino acid exchanges in the core region (M1-H5-M2), (2) truncation at the cytosolic C-terminus, and (3) deletions of putative promoter elements. While the core mutations W99C, N124K, and I142T led to significantly reduced macroscopic K(+) currents (1 to 8% of wild-type currents), the A103V and P110L variants retained substantial K(+) conductivity (23 and 35% of wild-type currents, respectively). Coexpression of A103V and P110L, resembling the compound heterozygous state of the affected individual, further reduced macroscopic currents to 9% of the wild-type currents. All mutants in the core region exerted a dominant-negative effect on wild-type ROMK1. The C-terminal frameshift (fs) mutation (H354fs) did not change current amplitudes compared with ROMK1 wild type, suggesting that a mechanism other than alteration of the electrophysiological properties may responsible for loss of channel activity. CONCLUSIONS: Analysis of ROMK mutants linked to HPS/aBS revealed a spectrum of mechanisms accounting for loss of channel function. Further characterization of the molecular defects might be helpful for the development of new therapeutic approaches.
Subject(s)
Bartter Syndrome/genetics , Mutation , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Prostaglandins E/metabolism , Animals , Bartter Syndrome/metabolism , Base Sequence , Child, Preschool , Codon, Nonsense , DNA Primers/genetics , Exons , Female , Genetic Linkage , Humans , In Vitro Techniques , Infant , Male , Mutation, Missense , Oocytes/metabolism , Pedigree , Potassium Channels/chemistry , Potassium Channels/metabolism , Sequence Deletion , Syndrome , Transfection , XenopusABSTRACT
We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K(+) channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1-24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.
Subject(s)
Genetic Linkage , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Protein Subunits , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cells, Cultured , Chromosomes, Human, Pair 17/genetics , Humans , Kidney/metabolism , Molecular Sequence Data , Oocytes/cytology , Oocytes/metabolism , Organ Specificity , Patch-Clamp Techniques , Potassium Channels/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Transfection , XenopusABSTRACT
Two cDNAs encoding novel K(+) channels, THIK-1 and THIK-2 (tandem pore domain halothane inhibited K(+) channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K(+) channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K(+) channel displaying weak inward rectification in symmetrical K(+) solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.
Subject(s)
Potassium Channels, Tandem Pore Domain , Potassium Channels/chemistry , Potassium Channels/genetics , Amino Acid Sequence , Anesthetics, Inhalation/pharmacology , Animals , Anions , Arachidonic Acid/pharmacology , Base Sequence , Brain/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 2 , Cloning, Molecular , DNA, Complementary/metabolism , Electrophysiology , Gastric Mucosa/metabolism , Green Fluorescent Proteins , Halothane/pharmacology , Humans , Hydrogen-Ion Concentration , In Situ Hybridization , Introns , Kidney/metabolism , Liver/metabolism , Luminescent Proteins/metabolism , Microscopy, Confocal , Molecular Sequence Data , Phylogeny , Potassium/metabolism , Potassium Channels/biosynthesis , Rats , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution , Xenopus/metabolismABSTRACT
The cDNAs for human and murine Receptor Activity Modifying Proteins and for the associated murine Calcitonin Receptor Like Receptor were isolated. The human RAMP1 and RAMP3 genes possess two introns and human RAMP2 possesses three introns. Human RAMP1 was assigned to chromosome 2q36-->q37.1, RAMP2 to 17q12-->q21.1 and RAMP3 to 7p13-->p12. Mouse Ramp1 was assigned to chromosome 1 and Ramp2 and Ramp3 were assigned to chromosome 11.
Subject(s)
Exons/genetics , Introns/genetics , Membrane Proteins/genetics , Physical Chromosome Mapping , Amino Acid Sequence , Animals , Chromosomes, Human, Pair 17/genetics , Chromosomes, Human, Pair 2/genetics , Chromosomes, Human, Pair 7/genetics , Cloning, Molecular , Databases as Topic , Expressed Sequence Tags , Humans , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , Radiation Hybrid Mapping , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Sequence AlignmentABSTRACT
The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.
Subject(s)
Adenosine Triphosphate/metabolism , Myocardium/metabolism , Potassium Channels/metabolism , Animals , Antimetabolites/pharmacology , Base Sequence , Capillaries/metabolism , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Endothelium, Vascular/metabolism , Guinea Pigs , In Vitro Techniques , Membrane Potentials/drug effects , Patch-Clamp Techniques , Potassium Channels/chemistry , Potassium Channels/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue DistributionABSTRACT
Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.
Subject(s)
Histidine/metabolism , Hydrogen-Ion Concentration , Potassium Channels, Tandem Pore Domain , Potassium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , Guinea Pigs , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Potassium Channels/metabolism , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , XenopusSubject(s)
Chromosomes, Human, Pair 19/genetics , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Amino Acid Sequence , Chromosome Mapping , Cloning, Molecular , DNA/chemistry , DNA/genetics , Exons , Genes/genetics , Humans , Hybrid Cells , Introns , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Capillary fragments were isolated from guinea pig hearts, and their electrical properties were studied using the perforated-patch and cell-attached mode of the patch-clamp technique. A voltage-dependent K(+) current was discovered that was activated at potentials positive to -20 mV and showed a sigmoid rising phase. For depolarizing voltage steps from -128 to +52 mV, the time to peak was 71 +/- 5 ms (mean +/- SE) and the amplitude of the current was 3.7 +/- 0.5 pA/pF in the presence of 5 mM external K(+). The time course of inactivation was exponential with a time constant of 7.2 +/- 0.5 s at +52 mV. The current was blocked by tetraethylammonium (inhibitory constant approximately 3 mM) but was not affected by charybdotoxin (1 microM) or apamin (1 microM). In the cell-attached mode, depolarization-activated single-channel currents were found that inactivated completely within 30 s; the single-channel conductance was 12.3 +/- 2.4 pS. The depolarization-activated K(+) current described here may play a role in membrane potential oscillations of the endothelium.
Subject(s)
Action Potentials , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Myocardium/cytology , Potassium/physiology , Animals , Electrophysiology , Guinea Pigs , In Vitro Techniques , Patch-Clamp Techniques , Potassium Channel BlockersABSTRACT
A simple method for analyzing the differential gene expression of coronary endothelial cells and cardiac muscle cells was developed. Cells were isolated from guinea pig hearts by collagenase digestion. In the diluted cell suspension, single cardiomyocytes and capillary fragments containing 6-15 endothelial cells could be identified morphologically. A simple "cell picker" was constructed using a polyethylene pipette with a tip diameter of approximately 150 micrometers that was attached to a micromanipulator and connected to an electric miniature valve. Intermittent suction pulses (1- to 2-cm water column) were applied by opening the valve for 100-200 ms at 1-s intervals. Cardiomyocytes (800-1,000) or capillary fragments (150) were picked under visual control using an inverted microscope. The cells were transferred to a reaction tube for RNA extraction, reverse transcription (RT), and DNA amplification (RT-PCR) with gene-specific and intron-spanning primers. All PCR products were verified by sequencing. Troponin T and endothelin-1 were found to be specific markers for guinea-pig cardiac muscle cells and coronary endothelial cells, respectively.
Subject(s)
Cell Separation/methods , Coronary Vessels/cytology , Endothelium, Vascular/cytology , Myocardium/cytology , Reverse Transcriptase Polymerase Chain Reaction , Animals , Coronary Vessels/metabolism , Endothelin-1/biosynthesis , Endothelium, Vascular/metabolism , Guinea Pigs , Myocardium/metabolism , Troponin/biosynthesisABSTRACT
Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.
Subject(s)
Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Potassium Channels/physiology , Potassium/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Animals, Newborn , Blotting, Northern , Brain/metabolism , Choroid Plexus/cytology , Choroid Plexus/metabolism , Cloning, Molecular , Epithelial Cells/metabolism , Humans , In Situ Hybridization , Molecular Sequence Data , Potassium Channels/metabolism , Rats , Rats, Wistar , Sequence Homology, Amino Acid , Tissue Distribution , Xenopus laevisABSTRACT
Loss of function mutations in kidney Kir1.1 (renal outer medullary potassium channel, KCNJ1) inwardly rectifying potassium channels can be found in patients suffering from hyperprostaglandin E syndrome (HPS), the antenatal form of Bartter syndrome. A novel mutation found in a sporadic case substitutes an asparagine by a positively charged lysine residue at amino acid position 124 in the extracellular M1-H5 linker region. When heterologously expressed in Xenopus oocytes and mammalian cells, current amplitudes from mutant Kir1.1a[N124K] channels were reduced by a factor of approximately 12 as compared with wild type. A lysine at the equivalent position is present in only one of the known Kir subunits, the newly identified Kir1.3, which is also poorly expressed in the recombinant system. When the lysine residue in guinea pig Kir1.3 (gpKir1.3) isolated from a genomic library was changed to an asparagine (reverse HPS mutation), mutant channels yielded macroscopic currents with amplitudes increased 6-fold. From single channel analysis it became apparent that the decrease in mutant Kir1.1 channels and the increase in mutant gpKir1.3 macroscopic currents were mainly due to the number of expressed functional channels. Coexpression experiments revealed a dominant-negative effect of Kir1.1a[N124K] and gpKir1.3 on macroscopic current amplitudes when coexpressed with wild type Kir1.1a and gpKir[K110N], respectively. Thus we postulate that in Kir1.3 channels the extracellular positively charged lysine is of crucial functional importance. The HPS phenotype in man can be explained by the lower expression of functional channels by the Kir1. 1a[N124K] mutant.
Subject(s)
Bartter Syndrome/genetics , Kidney Medulla/metabolism , Point Mutation , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Potassium Channels/physiology , Amino Acid Sequence , Animals , Bartter Syndrome/embryology , COS Cells , Cloning, Molecular , Female , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/physiology , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Potassium Channels/chemistry , Prostaglandins E/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Syndrome , Transfection , Xenopus laevisABSTRACT
To study the role of endothelial ATP-sensitive K+ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca2+]i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca2+]i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K+ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca2+]i followed by a sustained elevation of [Ca2+]i (19 of 40 experiments). [Ca2+]i in the endothelial cells increased from 32 +/- 7 nM at rest to 66 +/- 11 nM at the peak (n = 19). One third of the [Ca2+]i-transients showed irregular oscillations of [Ca2+]i. No significant difference in the [Ca2+]i-response induced by 100 nM or 1 muM diazoxide was found. Similar results were obtained with the K+ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca2+]i with fluorometric methods indicated that the hyperpolarization but not the [Ca2+]i-transient could be repeatedly induced in a single capillary by the K+ channel openers. Electrophysiological recordings of the membrane potential using the "perforated patch" method (n = 4), showed that rilmakalim (1 muM) induced hyperpolarization of capillaries towards the K+ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K+ channel openers induce [Ca2+]i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of KATP-channel in coronary capillaries characterised by high sensitivity to diazoxide.
Subject(s)
Calcium/metabolism , Capillaries/metabolism , Coronary Vessels , Potassium Channels/physiology , Animals , Capillaries/drug effects , Chromans/pharmacology , Diazoxide/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fluorescent Dyes , Fura-2 , Guinea Pigs , In Vitro Techniques , Ion Channel Gating/drug effects , Membrane Potentials , Potassium Channels/drug effects , Pyrrolidines/pharmacology , Spectrometry, FluorescenceABSTRACT
The novel weakly inward rectifying potassium channel Kir7.1 is a low-conductance channel that is predominantly expressed in epithelial cells. Here we describe a partial genomic characterization and the chromosomal assignment of the human Kir7.1 gene (KCNJ13). Analysis of the genomic structure using a PCR-based approach revealed a single 2088-bp intron in the coding region of KCNJ13. PCR analysis of monochromosomal and radiation hybrid panels assigns KCNJ13 to band 2q37 between markers D2S331 and D2S345. In addition, a single nucleotide polymorphism (C524-->T), leading to an exchange of a Thr with an Ile residue at amino acid position 175, was found.
Subject(s)
Bartter Syndrome/genetics , Chromosomes, Human, Pair 2 , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Heterozygote , Humans , Hybrid Cells , Molecular Sequence Data , Mutation , Physical Chromosome Mapping , Polymerase Chain Reaction , Polymorphism, GeneticABSTRACT
A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100-500 micrograms were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37 degrees C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 microM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 microM atractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 microM alpha-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 +/- 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells.
Subject(s)
Mitochondria, Heart/metabolism , Myocardium/metabolism , Oxidative Phosphorylation , Animals , Calorimetry/methods , Cell Membrane Permeability , Guinea Pigs , Hot Temperature , Mitochondria, Heart/ultrastructure , Myocardium/ultrastructureABSTRACT
1. The present study was designed to test if microvascular coronary endothelial cells express ATP-sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea-pig coronary capillaries equilibrated with the voltage-sensitive dye bis-oxonol (bis-[1,3-dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]). 2. The resting membrane potential of capillaries in physiological salt solution was -46 +/- 4.2 mV (n = 8) at room temperature (22 degrees C) as determined after calibration of the fluorescence using the Na(+)-K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10-20 mV amplitude were often observed. 3. A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nM to 1 microM), diazoxide (10 PM to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nM to 2 microM) reversed this hyperpolarization. 4. A glibenclamide-sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D-glucose (10 mM) from the superfusing solution or by substituting L-glucose for D-glucose. Maximum hyperpolarization was reached in less than 10 min. 5. Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.