Characterization of the ryanodine receptor/channel  of invertebrate muscle

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.

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Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene

Biochemical Journal ◽

10.1042/bj3420239 ◽

1999 ◽

Vol 342 (1) ◽

pp. 239-248 ◽

Cited By ~ 1

Author(s):

Nurit HADAD ◽

Wei FENG ◽

Varda SHOSHAN-BARMATZ

Keyword(s):

Ryanodine Receptor ◽

Single Channel ◽

Ruthenium Red ◽

Amino Group ◽

Channel Activity ◽

Closed Channel ◽

Release Channel ◽

Atp Binding Site ◽

Ph Values ◽

Stimulation Of

Modification of the ryanodine receptor (RyR)/Ca2+ release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t½ < 30 s) at 25 °C with low concentrations of DNFB [C50 (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding) = 5 μM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca2+ release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 °C); at pH values higher than 7.4 it reacted slowly (t½ = 20 min) with high concentrations of DNFB (C50 = 70 μM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca2+-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca2+. The DNFB-modified ryanodine-activated RyR channel showed fast transitions between open, closed and several sub-conductance states, and was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified closed channel or, if present during modification, prevented the inhibition of RyR channel activity by DNFB. Neither the stimulation nor the inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3′-O-(4-benzoyl)benzoyl-[α-32P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.

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

Physiological Genomics ◽

10.1152/physiolgenomics.00228.2005 ◽

2006 ◽

Vol 24 (3) ◽

pp. 290-297 ◽

Cited By ~ 10

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.

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Characterization of Single Channel Currents from Primary Cilia of Renal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m507793200 ◽

2005 ◽

Vol 280 (41) ◽

pp. 34718-34722 ◽

Cited By ~ 58

Author(s):

Malay K. Raychowdhury ◽

Margaret McLaughlin ◽

Arnolt J. Ramos ◽

Nicolás Montalbetti ◽

Richard Bouley ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilia ◽

Single Channel ◽

Renal Epithelial Cells ◽

Single Channel Currents ◽

Channel Currents

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Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

The Journal of General Physiology ◽

10.1085/jgp.113.2.177 ◽

1999 ◽

Vol 113 (2) ◽

pp. 177-186 ◽

Cited By ~ 102

Author(s):

Rafael Mejía-Alvarez ◽

Claudia Kettlun ◽

Eduardo Ríos ◽

Michael Stern ◽

Michael Fill

Keyword(s):

Lipid Bilayers ◽

Ryanodine Receptor ◽

Single Channel ◽

Monovalent Cation ◽

Planar Lipid Bilayers ◽

Physiological Conditions ◽

Single Channel Currents ◽

The Relationship ◽

Channel Currents ◽

Cardiac Ryanodine Receptor

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2–30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

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