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1.
Biomolecules ; 9(11)2019 10 25.
Article in English | MEDLINE | ID: mdl-31731488

ABSTRACT

The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These "mutation clusters" affect trafficking by different mechanisms, including protein stability.


Subject(s)
Genetic Predisposition to Disease , Potassium Channels, Inwardly Rectifying/genetics , Humans , Mutation , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/classification , Protein Transport , Syndrome
2.
Sci Rep ; 5: 15305, 2015 Oct 16.
Article in English | MEDLINE | ID: mdl-26470642

ABSTRACT

Potassium channels exhibit a modular design with distinct structural and functional domains; in particular, a highly conserved pore-loop sequence that determines their ionic selectivity. We now report the functional characterisation of a novel group of functionally non-selective members of the prokaryotic 'inward rectifier' subfamily of K(+) channels. These channels share all the key structural domains of eukaryotic and prokaryotic Kir/KirBac channels, but instead possess unique pore-loop selectivity filter sequences unrelated to any other known ionic selectivity filter. The strikingly unusual architecture of these 'NirBac' channels defines a new family of functionally non-selective ion channels, and also provides important insights into the modular design of ion channels, as well as the evolution of ionic selectivity within this superfamily of tetrameric cation channels.


Subject(s)
Bacterial Proteins/metabolism , Models, Molecular , Potassium Channels, Inwardly Rectifying/metabolism , Amino Acid Sequence , Bacterial Proteins/classification , Bacterial Proteins/genetics , Evolution, Molecular , Ion Transport , Molecular Sequence Data , Myxococcales/metabolism , Phylogeny , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/genetics , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
3.
Mol Pharmacol ; 74(5): 1333-44, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18723823

ABSTRACT

K(ATP) channels are metabolic sensors and targets of potassium channel openers (KCO; e.g., diazoxide and pinacidil). They comprise four sulfonylurea receptors (SUR) and four potassium channel subunits (Kir6) and are critical in regulating insulin secretion. Different SUR subtypes (SUR1, SUR2A, SUR2B) largely determine the metabolic sensitivities and the pharmacological profiles of K(ATP) channels. SUR1- but not SUR2-containing channels are highly sensitive to metabolic inhibition and diazoxide, whereas SUR2 channels are sensitive to pinacidil. It is generally believed that SUR1 and SUR2 are incompatible in channel coassembly. We used triple tandems, T1 and T2, each containing one SUR (SUR1 or SUR2A) and two Kir6.2Delta26 (last 26 residues are deleted) to examine the coassembly of different SUR. When T1 or T2 was expressed in Xenopus laevis oocytes, small whole-cell currents were activated by metabolic inhibition (induced by azide) plus a KCO (diazoxide for T1, pinacidil for T2). When coexpressed with any SUR subtype, the activated-currents were increased by 2- to 13-fold, indicating that different SUR can coassemble. Consistent with this, heteromeric SUR1+SUR2A channels were sensitive to azide, diazoxide, and pinacidil, and their single-channel burst duration was 2-fold longer than that of the T1 channels. Furthermore, SUR2A was coprecipitated with SUR1. Using whole-cell recording and immunostaining, heteromeric channels could also be detected when T1 and SUR2A were coexpressed in mammalian cells. Finally, the response of the SUR1+SUR2A channels to azide was found to be intermediate to those of the homomeric channels. Therefore, different SUR subtypes can coassemble into K(ATP) channels with distinct metabolic sensitivities and pharmacological profiles.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Receptors, Drug/metabolism , Animals , Blotting, Western , Cell Line , Chlorocebus aethiops , Humans , Immunohistochemistry , Immunoprecipitation , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/classification , Sulfonylurea Receptors , Xenopus laevis
4.
J Exp Biol ; 211(Pt 13): 2162-71, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18552306

ABSTRACT

A new member of the inward-rectifier K(+) channel subfamily Kir2 was isolated and characterised from the crucian carp (Carassius carassius) heart. When expressed in COS-1 cells this 422 amino acid protein produced an inward-rectifying channel with distinct single-channel conductance, mean open time and open probability. Phylogenetic sequence comparisons indicate that it is not homologous to any known vertebrate Kir channel, yet belongs to the Kir2 subfamily. This novel crucian carp channel increases the number of vertebrate Kir2 channels to five, and has therefore been designated as ccKir2.5 (cc for Carassius carassius). In addition to the ccKir2.5 channel, the ccKir2.2 and ccKir2.1 channels were expressed in the crucian carp heart, ccKir2.1 being present only in trace amounts (<0.8% of all Kir2 transcripts). Whole-cell patch clamp in COS-1 cells demonstrated that ccKir2.5 is a stronger rectifier than ccKir2.2 or ccKir2.1, and therefore passes weakly outward current. Single-channel conductance, mean open time and open probability of ccKir2.5 were, respectively, 1.6, 4.96 and 4.17 times as large as that of ccKir2.2. ccKir2.5 was abundantly expressed in atrium and ventricle of the heart and in skeletal muscle, but was a minor component of Kir2 in brain, liver, gill and kidney. Noticeably, ccKir2.5 was strongly responsive to chronic cold exposure. In fish reared at 4 degrees C for 4 weeks, ccKir2.5 mRNA formed 59.1+/-2.1% and 65.6+/-3.2% of all ccKir2 transcripts in atrium and ventricle, respectively, while in fish maintained at 18 degrees C the corresponding transcript levels were only 16.2+/-1.7% and 23.3+/-1.7%. The increased expression of ccKir2.5 at 4 degrees C occurred at the expense of ccKir2.2, which was the main Kir2 isoform in 18 degrees C acclimated fish. A cold-induced increase in the slope conductance of the ventricular I K1 from 707+/-49 to 1001+/-59 pS pF(-1) (P<0.05) was thus associated with an isoform shift from ccKir2.2 towards ccKir2.5, suggesting that ccKir2.5 is a cold-adapted and ccKir2.2 a warm-adapted isoform of the inward-rectifying K+ channel.


Subject(s)
Carps/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Acclimatization/genetics , Acclimatization/physiology , Animals , Base Sequence , COS Cells , Carps/genetics , Chlorocebus aethiops , Cold Temperature , DNA Primers/genetics , Electrophysiology , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Phylogeny , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tissue Distribution , Up-Regulation
5.
Gene ; 399(1): 46-52, 2007 Sep 01.
Article in English | MEDLINE | ID: mdl-17553638

ABSTRACT

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.


Subject(s)
Cichlids/genetics , Fish Proteins/genetics , Gene Duplication , Genetic Variation , Potassium Channels, Inwardly Rectifying/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Evolution, Molecular , Gene Dosage , Gene Expression , Genome , Molecular Sequence Data , Phylogeny , Potassium Channels, Inwardly Rectifying/classification , RNA, Messenger/analysis , RNA, Messenger/metabolism , Tissue Distribution
6.
Pflugers Arch ; 455(1): 83-95, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17520276

ABSTRACT

Phosphoinositides, such as phosphatidylinositol-bisphosphate (PIP(2)), control the activity of many ion channels in yet undefined ways. Inwardly, rectifying potassium (Kir) channels were the first shown to be dependent on direct interactions with phosphoinositides. Alterations in channel-PIP(2) interactions affect Kir single-channel gating behavior. Aberrations in channel-PIP(2) interactions can lead to human disease. As the activity of all Kir channels depends on their interactions with phosphoinositides, future research will aim to understand the molecular events that occur from phosphoinositide binding to channel gating. The determination of atomic resolution structures for several mammalian and bacterial Kir channels provides great promise towards this goal. We have mapped onto the three-dimensional channel structure the position of basic residues identified through mutagenesis studies that contribute to the sensitivity of a Kir channel to PIP(2). The localization of these putative PIP(2)-interacting residues relative to the channel's permeation pathway has given rise to a testable model, which could account for channel activation by PIP(2).


Subject(s)
Ion Channel Gating/physiology , Phosphatidylinositols/physiology , Potassium Channels, Inwardly Rectifying/physiology , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Phosphatidylinositol 4,5-Diphosphate/physiology , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/genetics
7.
Br J Pharmacol ; 149(5): 542-50, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17016513

ABSTRACT

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.


Subject(s)
Dihydropyridines/pharmacology , G Protein-Coupled Inwardly-Rectifying Potassium Channels/agonists , Myocytes, Smooth Muscle/drug effects , Urinary Bladder/drug effects , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/classification , Carbachol/pharmacology , Diazoxide/pharmacology , Dose-Response Relationship, Drug , Gliclazide/pharmacology , Glyburide/pharmacology , Humans , Immunochemistry , In Vitro Techniques , Membrane Potentials/drug effects , Muscle Relaxation/drug effects , Myocytes, Smooth Muscle/chemistry , Myocytes, Smooth Muscle/physiology , Patch-Clamp Techniques , Pinacidil/pharmacology , Potassium Channels/analysis , Potassium Channels/classification , Potassium Channels, Inwardly Rectifying/analysis , Potassium Channels, Inwardly Rectifying/classification , Receptors, Drug/analysis , Receptors, Drug/classification , Sulfonylurea Receptors , Urinary Bladder/cytology , Urinary Bladder/physiology
8.
J Cell Mol Med ; 10(1): 33-44, 2006.
Article in English | MEDLINE | ID: mdl-16563220

ABSTRACT

Glia in the central nervous system (CNS) express diverse inward rectifying potassium channels (Kir). The major function of Kir is in establishing the high potassium (K+) selectivity of the glial cell membrane and strongly negative resting membrane potential (RMP), which are characteristic physiological properties of glia. The classical property of Kir is that K+ flows inwards when the RMP is negative to the equilibrium potential for K+ (E(K)), but at more positive potentials outward currents are inhibited. This provides the driving force for glial uptake of K+ released during neuronal activity, by the processes of "K+ spatial buffering" and "K+ siphoning", considered a key function of astrocytes, the main glial cell type in the CNS. Glia express multiple Kir channel subtypes, which are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors, including pH, ATP, G-proteins, neurotransmitters and hormones. A feature of CNS glia is their specific expression of the Kir4.1 subtype, which is a major K+ conductance in glial cell membranes and has a key role in setting the glial RMP. It is proposed that Kir4.1 have a primary function in K+ regulation, both as homomeric channels and as heteromeric channels by co-assembly with Kir5.1 and probably Kir2.0 subtypes. Significantly, Kir4.1 are also expressed by oligodendrocytes, the myelin-forming cells of the CNS, and the genetic ablation of Kir4.1 results in severe hypomyelination. Hence, Kir, and in particular Kir4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction.


Subject(s)
Neuroglia/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Animals , Astrocytes/metabolism , Central Nervous System/cytology , Central Nervous System/metabolism , Mice , Mice, Knockout , Models, Biological , Neuroglia/physiology , Oligodendroglia/metabolism , Potassium/metabolism , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/physiology
11.
J Physiol ; 563(Pt 3): 725-44, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15618275

ABSTRACT

The strong inward rectification of the whole cell Kir2.1 current, which is very similar to the cardiac inward rectifier K(+) current (I(K1)), is caused by voltage-dependent blockade of outward currents by the intracellular polyamines spermine and spermidine. We recently showed that macroscopic Kir2.1 currents obtained from inside-out patches in the presence of various concentrations of cytoplasmic polyamines are well explained by the sum of the currents through two populations of channels that show differing susceptibilities to polyamine blockade. The outward currents obtained with 5-10 microM cytoplasmic spermine showed current-voltage relationships similar to those of I(K1) and were considered to flow mostly through a small population of channels exhibiting lower spermine sensitivity. Here we used inside-out patches to examine the blockade of macroscopic Kir2.1 currents by cytoplasmic Mg(2+) in the absence and presence of cytoplasmic spermine. Outward currents were blocked by 0.6 and 1.1 microM Mg(2+) in a concentration-dependent manner, but a small fraction ( approximately 0.1) of the macroscopic conductance was resistant to Mg(2+) at those concentrations, suggesting there are two populations of Kir2.1 channels with different sensitivities to Mg(2+). Furthermore, at those concentrations, Mg(2+) blocked inward currents by inducing a shallow blocked state that differed from the deeper state causing the inward rectification. In the presence of 1.1 microM Mg(2+) + 5 microM spermine, Mg(2+) blocked a substantial current component during depolarizing pulses and generated transient outward components, which is consistent with findings from earlier whole-cell experiments. In the steady state, Mg(2+) blocked the currents at voltages around and negative to the reversal potential and induced sustained outward components. The steady-state and time-dependent current amplitudes and the fractional blockades caused by spermine and Mg(2+) could be quantitatively explained by a model in which Mg(2+) competes with spermine to block the high-affinity channel and induces three conductance states. The present results suggest that the outward I(K1) flows through two populations of channels with different sensitivities to cytoplasmic blockers.


Subject(s)
Ion Channel Gating/physiology , Magnesium/pharmacology , Membrane Potentials/physiology , Myocytes, Cardiac/physiology , Polyamines/metabolism , Potassium Channels, Inwardly Rectifying/physiology , Spermine/pharmacology , Animals , Cell Line , Dose-Response Relationship, Drug , Humans , Ion Channel Gating/drug effects , Kidney/drug effects , Kidney/physiology , Membrane Potentials/drug effects , Mice , Models, Biological , Myocytes, Cardiac/drug effects , Polyamines/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/drug effects
12.
J Membr Biol ; 197(3): 179-91, 2004 Feb 01.
Article in English | MEDLINE | ID: mdl-15042349

ABSTRACT

Several inward rectifier K(+) (Kir) channels are inhibited by hypercapnic acidosis and may be involved in CO(2) central chemoreception. Among them are Kir1.1, Kir2.3, and Kir4.1. The Kir4.1 is expressed predominantly in the brainstem. Although its CO(2) sensitivity is low, coexpression of Kir4.1 with Kir5.1 in Xenopus oocytes greatly enhances the CO(2)/pH sensitivities of the heteromeric channels. If these Kir channels play a part in the central CO(2) chemosensitivity, they should be expressed in neurons of brainstem cardio-respiratory nuclei. To test this hypothesis, we performed in-situ hybridization experiments in which the expression of Kir1.1, Kir2.3, Kir4.1 and Kir5.1, and coexpression of Kir4.1 and Kir5.1 were studied in brainstem neurons using non-radioactive riboprobes. We found that mRNAs of these Kir channels were present in several brainstem nuclei, especially those involved in cardio-respiratory controls. Strong labeling was observed in the locus coeruleus, ventralateral medulla, parabrachial-Kölliker-Fuse nuclei, solitary tract nucleus, and area postrema. Strong expression was also seen in several cranial motor nuclei, including the nucleus of ambiguus, hypoglossal nucleus, facial nucleus and dorsal vagus motor nucleus. In general, the expression of Kir5.1 and Kir4.1 was much more prominent than that of Kir1.1 and Kir2.3 in all the nuclei. Evidence for the coexpression of Kir4.1 and Kir5.1 was found in a good number of neurons in these nuclei. The expression and coexpression of these CO(2)/pH-sensitive Kir channels suggest that they are likely to contribute to CO(2) chemosensitivity of the brainstem neurons.


Subject(s)
Brain Stem/metabolism , Carbon Dioxide/metabolism , Chemoreceptor Cells/metabolism , Gene Expression Regulation/physiology , Neurons/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Animals , Brain Stem/cytology , Culture Techniques , Female , Male , Neurons/cytology , Potassium Channels, Inwardly Rectifying/classification , Rats , Rats, Sprague-Dawley , Tissue Distribution
13.
Eur J Neurosci ; 18(8): 2110-8, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14622172

ABSTRACT

G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of Gi/o-coupled receptors. In central neurons, for example, they are activated by adenosine and GABA and, as such, they play an important role in neurotransmitter-mediated regulation of membrane excitability. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1-3.4 plus splice variants). In this study I have attempted to identify the channel subunits which contribute to the native GIRK current recorded from primary cultured rat hippocampal pyramidal neurons. Reverse transcriptase-polymerase chain reaction revealed the expression of mRNA for Kir3.1, 3.2A, 3.2C and 3.3 subunits and confocal immunofluorescence microscopy was used to investigate their expression patterns. Diffuse staining was observed on both cell somata and dendrites for Kir3.1 and Kir3.2A yet that for Kir3.2C was weaker and punctate. Whole-cell patch clamp recordings were used to record GIRK currents from hippocampal pyramidal neurons which were identified on the basis of inward rectification, dependence of reversal potential on external potassium concentration and sensitivity to tertiapin. The GIRK currents were enhanced by the stimulation of a number of Gi/o-coupled receptors and were inhibited by pertussis toxin. In order to ascertain which Kir3.x subunits were responsible for the native GIRK current I compared the properties with those of the cloned Kir3.1 + 3.2A and Kir3.1 + 3.2C channels heterologously expressed in HEK293 cells.


Subject(s)
GTP-Binding Proteins/physiology , Hippocampus/cytology , Neurons/physiology , Potassium Channels, Inwardly Rectifying/physiology , Potassium Channels/physiology , Adenosine/pharmacology , Analgesics/pharmacology , Animals , Animals, Newborn , Baclofen/pharmacology , Benzoxazines , Blotting, Northern , Calcium Channel Blockers/pharmacology , Carbachol/pharmacology , Cell Cycle Proteins/metabolism , Cells, Cultured , Cholinergic Agonists/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Embryo, Mammalian , Free Radical Scavengers/pharmacology , G Protein-Coupled Inwardly-Rectifying Potassium Channels , GABA Agonists/pharmacology , Hormones/pharmacology , Humans , Kidney , Membrane Potentials/drug effects , Microscopy, Confocal/instrumentation , Microscopy, Confocal/methods , Morpholines/pharmacology , Naphthalenes/pharmacology , Neurons/drug effects , Patch-Clamp Techniques/methods , Pertussis Toxin/pharmacology , Potassium Channels/metabolism , Potassium Channels, Inwardly Rectifying/classification , Protein Subunits/physiology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Serotonin/pharmacology , Somatostatin/pharmacology , Time Factors
14.
Biophys J ; 84(4): 2345-56, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12668443

ABSTRACT

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels.


Subject(s)
Cell Membrane Permeability , Ion Channel Gating , Lipid Bilayers/chemistry , Models, Molecular , Phosphatidylcholines/chemistry , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/classification , Animals , Computer Simulation , Humans , Mammals , Membrane Fluidity , Porosity , Potassium Channels, Inwardly Rectifying/genetics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/genetics , Reproducibility of Results , Sensitivity and Specificity , Structure-Activity Relationship , Water/chemistry
15.
Ophthalmic Res ; 34(6): 343-8, 2002.
Article in English | MEDLINE | ID: mdl-12483021

ABSTRACT

Ion channels were studied using the whole-cell patch clamp technique in bovine retinal and choroidal microvascular endothelial cells (MVEC) cultured under the same conditions. The two types of MVEC expressed inward currents at hyperpolarizing voltage steps and showed small outward currents at depolarizing steps. The extrapolated reversal potentials of the inward currents were near to the potassium equilibrium potential. Cs(+) and the K(+) channel blocker TEA reduced the amplitudes of the currents indicating the selectivity and permeability for potassium. This was confirmed by changes of outside K(+) concentration shifting the I-V curves to the right. RT-PCR studies revealed the presence of mRNA of Kir2.1, an inwardly rectifying K(+) channel, in retinal and choroidal MVEC. The profile of the small outward currents is related to the Kv family but not identical with the Kv1.4 subtype.


Subject(s)
Choroid/cytology , Endothelium, Vascular/metabolism , Potassium Channels, Inwardly Rectifying/isolation & purification , Retina/cytology , Animals , Cattle , Cells, Cultured , Cesium/pharmacology , DNA, Complementary/analysis , Endothelium, Vascular/physiology , Fluorescent Antibody Technique , Lipoproteins, LDL , Membrane Potentials/drug effects , Membrane Potentials/physiology , Patch-Clamp Techniques/methods , Potassium/metabolism , Potassium/pharmacology , Potassium Channels, Inwardly Rectifying/classification , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/physiology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Tetraethylammonium Compounds/pharmacology
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