Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum |
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Authors: | Eric Rousseau Hélène Chabot Catherine Beaudry Bernard Muller |
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Institution: | (1) Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, J1H 5N4 Sherbrooke, Quebec, Canada;(2) CNRS - URA 600, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch, France |
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Abstract: | In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K+), and the presence of short and long lasting subconducting states. Its open probability Po increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca2+ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K+ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of Ca2+] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca2+, Ba2+ or Mg2+-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca2+ while its Ca2+ sensitivity was potentiated by millimolar ATP. However Mg2+ and calmodulin modulated its gating behavior. Ca2+ releasing drugs such as caffeine and ryanodine increased its Po. All these features are characteristics of the SR Ca2+ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca2+ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane. |
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Keywords: | cardiac sarcoplasmic reticulum K+ channel Ca2+ release channel |
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