Abstract
Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.
Highlights
Transverse tubule (TmT)embrane vesicleshave been isolated from the skeletaml uscle of normal and malignant hyperthermia-susceptible (MHS) pigs
The characteristic staining pattern of electrophoretically fractionated sarcoplasmic reticulum (SR) with Stains All has been used previously to identify the M, 60,000 proteincalsequestrin andthe M, 100,000 Ca2'-ATPase in SR [27, 32]. While these two bands were clearly visible in pig SR,no staining of pig transverse tubule (TT) protein by Stains All was detected.The M, 60,000 component was detectable when as little as 1.5 pg of SR protein was loaded per lane, suggesting the SR content of the porcine TT vesicles was less than 4% and notsignificantly different between normal and MHSTT preparations
0.00 0 to a similar extent (30-36%), such that [3H]nitrendipine binding to MHS TT was still only 53% of that measured in normal TT (Table V)
Summary
MH as previously described [7, 9]. Sodium thiamylal was purchased concentration between 0.9 and 1.1mg/ml, in the presence of 5 nM from Bio-Ceutic Laboratories, St. [3H]D888binding to TT vesicles (10pg) was measured in a medium unlabeled nitrendipine was the gift of Miles Laboratories, Inc., New (1.0 ml final volume) containing 50 mM Tris-C1 buffer (pH 7.5) and. Preparation of TT Vesicles-While pigs were anesthetizedwith 0.05% polyethyleneimine [26] and washed with 16 ml of ice-cold 200 thiamylal and mechanically ventilated, the longissimus dorsi muscle mM choline chloride, 20 mM Tris-C1 buffer (pH 7.5). Muscle microsomes used for the were heated to 90 "C for 10 min, mM dithiothreitol was added, and isolation of TT vesicles were prepared according to Fernandez et al the samples were electrophoresed on 5-20% gradient polyacryl-. S.E. and were analyzed by a Student's sucrose, 20 mM Tris maleate buffer (pH 7.0), and the final protein t test.
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