Identification and characterization of a novel member of the ATP-sensitive K+ channel subunit family, Kir6.3, in zebrafish

2006 ◽  
Vol 24 (3) ◽  
pp. 290-297 ◽  
Author(s):  
Changliang Zhang ◽  
Takashi Miki ◽  
Tadao Shibasaki ◽  
Masaaki Yokokura ◽  
Atsunori Saraya ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Channel ◽  
Chimeric Protein ◽  
Microscopic Analysis ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
Er Retention ◽  
Channel Currents

ATP-sensitive K+ (KATP) channels play a crucial role in coupling cellular metabolism to membrane potential. In addition to the orthologs corresponding to Kir6.1 and Kir6.2 of mammals, we have identified a novel member, designated Kir6.3 (zKir6.3), of the inward rectifier K+ channel subfamily Kir6.x in zebrafish. zKir6.3 is a protein of 432 amino acids that shares 66% identity with mammalian Kir6.2 but differs considerably from mammalian Kir6.1 and Kir6.2 in the COOH terminus, which contain an Arg-Lys-Arg (RKR) motif, an endoplasmic reticulum (ER) retention signal. Single-channel recordings of reconstituted channels show that zKir6.3 requires the sulfonylurea receptor 1 (SUR1) subunit to produce KATP channel currents with single-channel conductance of 57.5 pS. Confocal microscopic analysis shows that zebrafish Kir6.3 requires the SUR1 subunit for its trafficking to the plasma membrane. Analyses of chimeric protein between human Kir6.2 and zKir6.3 and a COOH-terminal deletion of zKir6.3 indicate that interaction between the COOH terminus of zKir6.3 and SUR1 is critical for both channel activity and trafficking to the plasma membrane. We also identified zebrafish orthologs corresponding to mammalian SUR1 (zSUR1) and SUR2 (zSUR2) by the genomic database. Both Kir6.3 and SUR1 are expressed in embryonic brain of zebrafish, as assessed by whole mount in situ hybridization. These data indicate that Kir6.3 and SUR1 form functional KATP channels at the plasma membrane in zebrafish through a mechanism independent from ER retention by the RKR motif.

Characterization of the ryanodine receptor/channel of invertebrate muscle

1998 ◽  
Vol 274 (2) ◽  
pp. R494-R502 ◽  
Author(s):  
Kerry E. Quinn ◽  
Loriana Castellani ◽  
Karol Ondrias ◽  
Barbara E. Ehrlich
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Microscopic Analysis ◽  
Ruthenium Red ◽  
Electron Microscopic ◽  
Release Channel ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Bell Shaped Curve

Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161–173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM–10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner. J. Biol. Chem. 267: 15893–15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.

scholarly journals Isolation and characterization of the urothelial lumenal plasma membrane.

1977 ◽  
Vol 73 (2) ◽  
pp. 382-399 ◽  
Author(s):  
J S Caruthers ◽  
M A Bonneville
Keyword(s):  
Plasma Membrane ◽  
Sodium Dodecyl ◽  
Modified Technique ◽  
Sialic Acid Content ◽  
Polarity Index ◽  
Total Sialic Acid ◽  
Isolation And Characterization ◽  
A Value

The lumenal plasma membrane has been isolated from transitional epithelial cells (urothelium) lining the urinary bladder in sheep by a modified technique involving treatment with hypotonic thioglycolate. The isolated membranes, like those in situ, are distinguished morphologically by arrays of hexagonal particles (in plague regions) separated by smooth interplaque regions. These plaque regions, specifically, can be isolated from the lumenal plasma membrane. Of the proteins constituting the lumenal plasma membrane, five were found to characterize the plaque regions and, in particular, the 33,000-dalton species appears to be most heavily concentrated in the sodium dodecyl sulfate-polyacrylamide gel pattern of the isolated plaque regions. Lipid analyses showed that there are approximately 0.93 mg of phospholipid and 0.27 mg of cholesterol for each milligram of protein, giving a value of 55% lipids and 45% proteins for the composition of the lumenal plasma membrane. The total sialic acid content was measured to be approximately 0.038 micronmol/mg protein for the plasma membrane. Several plasma membrane marker enzymes were found to be associated with the lumenal plasma membrane fraction, but only the 5'-nucleotidase activity was found to be further enriched in the plaque region fraction. Amino acid analysis of the intrinsic proteins of the plaques indicated a polarity index of 45%.

scholarly journals Characterization of the pancreatic type ATP-sensitive K+ channel with mutations in the nucleotide-binding fold of the sulfonylurea receptor

1997 ◽  
Vol 73 ◽  
pp. 154
Author(s):  
Vez P Repunte ◽  
Mitsuhiko Yamada ◽  
Yoshiyuki Horio ◽  
Shojiro Isomoto ◽  
Yoshihisa Kurachi
Keyword(s):  
Nucleotide Binding ◽  
K Channel ◽  
Sulfonylurea Receptor

scholarly journals Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver

1988 ◽  
Vol 252 (2) ◽  
pp. 437-445 ◽  
Author(s):  
J M Graham ◽  
D J Winterbourne
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Heparan Sulphate ◽  
Membrane Vesicles ◽  
Membrane System ◽  
Glycoprotein Synthesis ◽  
Golgi Membranes ◽  
Two Populations

We report on the incorporation of radiolabelled sulphate into proteoglycan in the ‘in situ’-perfused rat liver. After 5 min virtually all of the [35S]sulphate was incorporated into heparan sulphate; no partially sulphated precursors were detected. Pulse-chase experiments, followed by centrifugation in gradients of sucrose and metrizamide, showed that, at 5 min, the heparan sulphate was associated predominantly with the Golgi membranes. Over the next 20 min, intact proteoglycan appeared at the plasma membrane. At intermediate times the heparan sulphate was detected simultaneously in two distinct populations of membrane vesicles. Whether the heparan sulphate in these two populations has two different destinies (e.g. plasma membrane or secretion) is not yet clear. Subfractionation of the Golgi membranes showed that the N-sulphotransferase co-purified with the heparan [35S]sulphate and was separable from the galactosyltransferase of glycoprotein synthesis, confirming that the Golgi membrane system is functionally segregated. Subfractionation also permitted an almost 100-fold purification of the N-sulphotransferase over the homogenate: this will provide an excellent starting material for isolation and further characterization of the enzyme.

scholarly journals Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies.

1992 ◽  
Vol 172 (1) ◽  
pp. 271-287
Author(s):  
A Bertl ◽  
C L Slayman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Single Channel ◽  
K Channel ◽  
Substrate Uptake ◽  
Alkaline Ph ◽  
Open Probability ◽  
Extracellular Medium ◽  
Yeast Saccharomyces Cerevisiae ◽  
Charge Balancing

Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.

Characterization of single non-inactivating potassium channels in primary neuronal cultures of Drosophila

1989 ◽  
Vol 145 (1) ◽  
pp. 173-184
Author(s):  
D. Yamamoto ◽  
N. Suzuki
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Neuronal Cultures ◽  
Patch Clamp Technique ◽  
Primary Neuronal Cultures ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cytoplasmic Side

Permeability and gating properties of single, non-inactivating, K+ channel currents in cultured Drosophila neurons were studied using the gigaohm-seal patch-clamp technique. The non-inactivating K+ currents were activated by depolarizing the membrane to −30 mV or to more positive potentials. The slope conductance of the channel was estimated to be 17.6 +/− 3.70 pS when the cytoplasmic side of the inside-out membrane patch was perfused with solutions containing 145 mmoll-1 K+. The single-channel conductance was temperature-sensitive, with a Q10 of 1.44 between 10 and 20 degrees C. Single-channel currents could be recorded when the cytoplasmic K+ was replaced with NH4+, Rb+ or Na+, but not with Cs+. The conductance ratio of the channel for these cations was: K+ (1) greater than NH4+(0.53) greater than Rb+ (0.47) greater than Na+ (0.44). Tetraethylammonium (TEA+) ions applied at a concentration of 10 mmoll-1 to the cytoplasmic side of the membrane increased the frequency of ‘blank’ traces which contained no channel openings during repetitive depolarization. In addition, single-channel amplitude was reduced by about 20%. The open-time distribution was fitted by a single exponential function, whereas the closed-time distribution required a three-exponential fit. Permeability and gating properties of single, non-inactivating K+ channel currents in neurons of eag, a mutant which has defects in the delayed rectifier K+ channel, were indistinguishable from those recorded from wild-type neurons.

scholarly journals Characterization of Ether-à-go-go Channels Present in Photoreceptors Reveals Similarity to IKx, a K+ Current in Rod Inner Segments

1998 ◽  
Vol 111 (4) ◽  
pp. 583-599 ◽  
Author(s):  
Stephan Frings ◽  
Nicole Brüll ◽  
Claudia Dzeja ◽  
Albert Angele ◽  
Volker Hagen ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Splice Variants ◽  
K Channel ◽  
Retinal Ganglion ◽  
Activation Kinetics ◽  
Rod Photoreceptors ◽  
Transmembrane Segments ◽  
K Current

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.

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