It is thought that in skeletal muscle excitation-contraction (EC) coupling, the release of Ca2+ from the sarcoplasmic reticulum is controlled by the dihydropyridine (DHP) receptor in the transverse tubular membrane, where it serves as the voltage sensor. We have shown previously that injection of an expression plasmid carrying the skeletal muscle DHP receptor complementary DNA restores EC coupling and L-type calcium current that are missing in skeletal muscle myotubes from mutant mice with muscular dysgenesis. This restored coupling resembles normal skeletal muscle EC coupling, which does not require entry of extracellular Ca2+. By contrast, injection into dysgenic myotubes of an expression plasmid carrying the cardiac DHP receptor cDNA produces L-type calcium current and cardiac-type EC coupling, which does require entry of extracellular Ca2+. To identify the regions responsible for this important functional difference between the two structurally similar DHP receptors, we have expressed various chimaeric DHP receptor cDNAs in dysgenic myotubes. The results obtained indicate that the putative cytoplasmic region between repeats II and III of the skeletal muscle DHP receptor is an important determinant of skeletal-type EC coupling.

The skeletal muscle dihydropyridine (DHP) receptor is essential in excitation-contraction (EC) coupling. The receptor is postulated to be the voltage sensor giving rise to the intramembrane current, termed charge movement. We have now tested this hypothesis using myotubes from mice with the muscular dysgenesis mutation, which alters the skeletal muscle DHP receptor gene and prevents its expression. Our results indicate that charge movement is deficient in dysgenic myotubes but is fully restored following injection of an expression plasmid carrying the rabbit skeletal muscle DHP receptor complementary DNA, strongly supporting the hypothesis that the DHP receptor is the voltage sensor for EC coupling in skeletal muscle. Additionally, our data obtained for normal and chimaeric DHP receptor constructs demonstrate that DHP receptors with widely differing abilities to function as calcium channels and to mediate EC coupling produce very similar charge movements.

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2؉ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca 2؉ through the channel is not required for skeletal muscletype EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser 687 of the skeletal II-III loop has been reported to be rapidly phosphorylated in vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alaninesubstituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725-742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725-742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711-765). However, residues 725-742 appeared to be critical because skeletaltype coupling was not produced either by a chimera with skeletal residues 711-732 or by one with skeletal residues 734 -765. Dihydropyridine receptors (DHPRs)1 in skeletal and cardiac muscle are closely related proteins encoded by two different genes (1, 2). In both muscle types, DHPRs serve dual functions (2-6) as voltage-gated, L-type Ca 2ϩ channels and as a trigger for excitation-contraction (EC) coupling, which controls the release of Ca 2ϩ through ryanodine receptors (RyRs) (7) in the sarcoplasmic reticulum. However, the mechanism of EC coupling is different in cardiac and skeletal muscle. In cardiac muscle, depolarization causes opening of L-type Ca 2ϩ channels, and the resulting entry of extracellular Ca 2ϩ triggers RyRs to release Ca 2ϩ (6). In skeletal muscle, EC coupling does not require Ca 2ϩ entry (8); rather, depolarization causes some other kind of signal to be transmitted from DHPRs to RyRs. To identify regions that are critical for this skeletal-type EC coupling, we have previously used the approach of constructing cDNAs encoding chimeras of the skeletal and cardiac DHPRs (3). Expression of these chimeric DHPRs in dysgenic myotubes, which lack a functional gene for the skeletal DHPR (9), revealed that the putative cytoplasmic region between repeats II and III (II-III loop, amino acids 666 -791) of the skeletal DHPR is an important determinant of skeletal-type EC coupling (3).Comparison of the skeletal and cardiac DHPRs reveals differences scattered throughout the II-III loops. One important difference is the presence of a site (Ser 687 ) in the skeletal II-III loop, which is phosphorylated by cyclic AMP-dependent protein kinase (PKA) (1, 10) and is lacking in t...

Membrane depolarization causes many kinds of ion channels to open, a process termed activation. For both Na+ channels and Ca2+ channels, kinetic analysis of current has suggested that during activation the channel undergoes several conformational changes before reaching the open state. Structurally, these channels share a common motif: the central element is a large polypeptide with four repeating units of homology (repeats I-IV), each containing a voltage-sensing region, the S4 segment. This suggests that the distinct conformational transitions inferred from kinetic analysis may be equated with conformational changes of the individual structural repeats. To investigate the molecular basis of channel activation, we constructed complementary DNAs encoding chimaeric Ca2+ channels in which one or more of the four repeats of the skeletal muscle dihydropyridine receptor are replaced by the corresponding repeats derived from the cardiac dihydropyridine receptor. We report here that repeat I determines whether the chimaeric Ca2+ channel shows slow (skeletal muscle-like) or rapid (cardiac-like) activation.

Each ofthe four repeats (or motifs) ofvoltagegated Ion ch ls I thought to contaIn six tran1 membrane segments (S1-S6 (5,6) myotubes were prepared from newborn mice essentially following described procedures (5,7,8). Briefly, myoblasts isolated from fore-and hindlimbs were plated at a density of 2 x 104 per 35-mm Falcon Primaria dish, on which a grid of -2-mm squares was ruled. After fusion of myoblasts into myotubes had begun, some cultures were treated for 24 hr with 10 pM cytosine arabinoside. The cultures were used within 6-9 days after plating cells.Consuction of DHPR cDNAs. To construct pCAC7, the nucleotides AAG (residues 493-495) of pCAC6 (8), the expression plasmid of the rabbit skeletal muscle DHPR used in our previous studies (2,(8)(9)(10)(11), were replaced with the nucleotides CGC by site-directed mutagenesis (12). The compositions of the individual chimeric DHPRs are given below [Sk and Ca, skeletal muscle (13) and cardiac (14) DHPR, respectively; numbers in parentheses are amino acid numbers; for a region of identical sequence that flanks the joining site between the cardiac and skeletal muscle DHPRs, amino acid numbers are given as if that entire region were of cardiac sequence]: SkC15 (2) Sk

This study was designed to examine the interrelationships between performance in endurance running events from 10 to 90 km, training volume 3-5 weeks prior to competition, and the fractional utilization of maximal aerobic capacity (%VO2max) during each of the events. Thirty male subjects underwent horizontal treadmill testing to determine their VO2max, and steady-state VO2 at specific speeds to allow for calculation of %VO2max sustained during competition. Runners were divided into groups of ten according to their weekly training distance (group A trained less than 60 km X week-1, group B 60 to 100 km X week-1, and group C more than 100 km X week-1). Runners training more than 100 km X week-1 had significantly faster running times (average 19.2%) in all events than did those training less than 100 km X week-1. VO2max or %VO2max sustained during competition was not different between groups. The faster running speed of the more trained runners, running at the same %VO2max during competition, was due to their superior running economy (19.9%). Thus all of the group differences in running performance could be explained on the basis of their differences in running economy. These findings suggest either that the main effect of training more than 100 km X week-1 may be to increase running economy, or that runners who train more than 100 km X week-1 may have inherited superior running economy.(ABSTRACT TRUNCATED AT 250 WORDS)

The whole-cell patch-clamp technique was used to study voltagedependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed Ic~,t,, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, Ic~ is usually so small that it cannot be detected. In addition, Ic~ay, is apparently absent from normal skeletal muscle. From a holding potential of -80 mV, Ic~.,a becomes apparent for test pulses to ~-20 mV and peaks at ~ + 20 mV. The current activates rapidly (rise time ~5 ms at 20~ and with 10 mM Ca as charge cattier inactivates little or not at all during a 200-ms test pulse. Thus, Ic~.,~ activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current.

To prevent thermal injuries during distance running, the American College of Sports Medicine proposes that between 0.83 and 1.65 l of water should be ingested each hour during prolonged exercise. Yet such high rates of fluid intake have been reported to cause water intoxication. To establish the freely-chosen rates of fluid intake during prolonged competitive exercise, we measured fluid intake during, body weight before and after, and rectal temperature after competition in a total of 102 runners and 91 canoeists competing in events lasting from 170-340 min. Fluid intakes during competition ranged from 0.29-0.62 l.h-1; rates of water loss ranged from 0.69-1.27 l.h-1 in the runners; values were lower in the canoeists. Mean post-race rectal temperatures ranged from 38.0-39.0 degrees C. There was no relationship between the degree of dehydration and post-race rectal temperature. We conclude that hyperthermia is uncommon in prolonged competitive events held in mild environmental conditions, and that exercise intensity, not the level of dehydration, is probably the most important factor determining the postexercise rectal temperature. During prolonged exercise in mild environmental conditions, a fluid intake of 0.5 l.h-1 will prevent significant dehydration in the majority of athletes.