Acta Physiologica 2012; Volume 205, Supplement 690
Joint Meeting of the Hungarian Biophysical Society, Hungarian Physiological Society, Hungarian Society of Anatomists and Hungarian Society of Microcirculation & Vascular Biology
ALTERED MODULATION OF THE SKELETAL TYPE RYANODINE RECEPTOR / CALCIUM RELEASE CHANNEL BY ATP IN MALIGNANT HYPERTHERMIA
Abstract number: L9
Sarkozi1 S, Lukacs1 B, Bardi1 M, Jona1 I
1Research Centre for Molecular Medicine & Department of Physiology, University of Debrecen, Health Science Centre, Debrecen, Hungary
We have shown previously that the activity of the Ryanodine Receptor / Calcium release channel (RyR/CRC) is modulated by ATP. Increasing ATP concentration results in an increase of open probability of the RyR/CRC and the dose response curve of the phenomena is biphasic having two distinctive activatory processes. The amplitude ratio of the two steps are 1:3 suggesting strong cooperation between the ATP binding sites and independent but cooperative binding of the ATP on the RyR1 monomers. It was also shown by several laboratories that mutations leading to malignant hyperthermia (MH) also increases the calcium sensitivity of the channel. Our aim was to test whether the ATP pharmacology is affected in MH and if this is the case in what way.
Heavy SR vesicle was prepared as described previously using longissimus dorsi of a swine (Pietrin strain), which carries an MH causing homozygous arg615cys mutation. Following CHAPS+lipid solubilization, the functional RyR1 tetramer - the channel complex - was incorporated into a lipid bilayer. The bathing medium contained symmetrical 250 mM KCl 20mM PIPES pH:7.4. Free (ionized) calcium concentration was established using Ca-EGTA calcium buffer, calculated by Fabiato's method. Under voltage clamp conditions the channel current was recorded and the channel parameters were determined: such as open probability (Po), mean open time and specific conductance. The ATP pharmacology of the RyR/CRC was determined using 50 mM Ca2+ free trans and 472 nM Ca2+ free cis, applying increasing Na2ATP concentration on the cis side.
The mutant channel showed higher open probability compared to the wild type even in the absence of ATP. The mean open time was slightly higher in the mutant, but not significantly different from the wt. The ATP pharmacology of the mutant channel was different from the wt: the pronounced two phases disappeared from the ATP dependence of the open probability function. The increase of the open probability has two components: the mean open time increased significantly above 100 mM, and the number of open events increased even more pronounced above 150 mM ATP. The majority of the Po increase was attributed to the increase of the number of open events. All point histograms showed clearly two peaks without a trace of subconductance state: meaning that the synchrony of the four RyR1 monomers has not been changed due to the given mutation.
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Acta Physiologica 2012; Volume 205, Supplement 690 :L9