[49] Planar bilayer recording of ryanodine receptors of sarcoplasmic reticulum
Publisher Summary
This chapter describes methods to incorporate sarcoplasmic reticulum (SR) Ca2+ channels, also called “ryanodine receptors,” into planar bilayers. Recordings of ryanodine receptors are best made using CsCI as current carrier, instead of the Ca-HEPES and Tris-HEPES solutions. The use of CsCI instead of Ca-HEPES and Tris-HEPES eliminate the need for perfusion and the need for large and unphysiological gradients of Ca2+, which severely inactivate the channel. SR Cl- channels can be separated from ryanodine receptors based on reversal potential. The polarity of channels incorporated into the bilayer is constant, in the majority of cases. The myoplasmic end of the receptor faces into the cis solution, and the intravesicular end faces into the trans solution. Polarity can be easily confirmed by the cis-activation of channels by adenosine triphosphate (ATP) and micromolar Ca2+, which are myoplasmic activators of ryanodine receptors.
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Cited by (28)
Structural and Molecular Bases of Sarcoplasmic Reticulum Ion Channel Function
2014, Cardiac Electrophysiology: From Cell to Bedside: Sixth EditionRyanodine receptors/Calcium release channels in heart failure and sudden cardiac death
2001, Journal of Molecular and Cellular CardiologyCalcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+homeostasis in the heart is beginning to emerge. The intracellular Ca2+release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.
Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings
1999, Biochimica et Biophysica Acta - Molecular Cell ResearchThe actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (Po) of the calcium release channel at activating calcium concentrations (20–100 μM Ca2+) by 60–100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6–2 mM) decreased Po. Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced Po at activating Ca2+ (20–100 μM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca2+ (0.6 μM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca2+. High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca2+. The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.
Reconstitution of native and cloned channels into planar bilayers
1999, Methods in EnzymologyA planar bilayer is an artificial membrane formed across a small hole, ∼50 μm or larger in diameter. The hole on which the membrane is formed is usually placed in a thin plastic partition separating two aqueous compartments, but artificial bilayers may also be formed on a glass micropipette tip. Insertion or incorporation of a channel-forming molecule into such a membrane provides a simple experimental system for electrical recording of channel-mediated currents. Planar bilayer recording of ion channels is practiced for a number of reasons. It is a technique that yields incredibly rich mechanistic information on a relatively low budget while offering kaleidoscopic displays of single-channel fluctuations that some workers find delightful, even soothing.. This chapter highlights technical aspects of selected examples where the bilayer approach has been widely applied to functional analysis of major classes of ion channel proteins. Identification of techniques and preparations that have been found to be most generally applicable may facilitate extension of the bilayer approach to other classes of channel proteins that have not yet been thoroughly domesticated.
Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca<sup>2+</sup> affinity of regulatory Ca<sup>2+</sup> sites in single channel recordings and [<sup>3</sup>H]ryanodine binding
1998, Biochimica et Biophysica Acta - Molecular Cell ResearchThe actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid (p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20–100 μM) increased the mean open probability (Po) of the calcium channel at subactivating (20 nM), maximally activating (20–100 μM) and inhibitory (1–4 mM) Ca2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 μM 4-CMPS caused a transient increase in Po, followed by a low activity state within 1 min. At inhibitory Ca2+ concentrations Po was increased to values observed with maximally activating Ca2+ or lower, inhibitory Ca2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [3H]Ryanodine binding was increased up to four-fold with 3–15 μM 4-CMPS/p-CMB (Hill coefficient 1.7–2.0) at 4 μM Ca2+ and reduced at high concentrations (50–200 μM). The increase in [3H]ryanodine binding by 10 μM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 μM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.
Intercellular calcium signaling via gap junction in connexin-43- transfected cells
1998, Journal of Biological ChemistryIn excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells.
We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.