1. A method for producing rapid [Ca2+] and [Sr2+] changes in the frog skinned muscle fibre preparation while maintaining constant all other cationic concentrations (Moisescu, 1976a, b) is described and analysed in detail. 2. Different experiments, some of them involving the Ca2+‐sensitive photoprotein aequorin, as well as theoretical considerations, indicate that with this method one can produce a Ca2+ (or Sr2+) concentration change within 0.1‐‐0.15 sec in a whole preparation having a diameter of 50 micrometer. 3. The rate of force development was similar to that observed in vivo. 4. The radial diffusion coefficient of EGTA in relaxed myofibrillar preparations was measured and found to be 4.6 x 10(‐6) cm2sec‐1 at 20 degrees C. 5. The sarcoplasmic reticulum in myofibrillar bundles was found to be active with respect to both Ca2+ and Sr2+ in the solutions used ([Mg2+] 1 mM; [Na] 30 mM; [K] 140‐170 mM; [Cl] less than or equal to 20 mM; pH 7.10). 6. The amount of Ca released by caffeine from internal stores (previously loaded with Ca) can raise the total Ca concentration in the muscle fibre preparation by at least 1.8 mM. 7. The presence of 10 mM‐caffeine in all bathing solutions reduced drastically the ability of the sarcoplasmic reticulum to accumulate both Ca and Sr.

Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.

The RyR2 channel leak under diastolic conditions could cause SR-Ca2+ depletion, concomitantly arrhythmogenesis and heart failure in a subgroup of ARVC patients of genotype T4. A change in the RyR2 subunit composition due to the combined expression of both SNPs alters the behaviour of the tetrameric channel complex.

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The total membrane concentrations of PtdIns, PtdIns4P, and PtdIns(4,5)P, contribute to the functional capacity of the Ins(1,4,5)Pj signalling system which is operating in skeletal muscle but the function of which is still unknown. Total amounts of these phosphoinositides have been determined in purified membranes of transverse tubules (lT) and terminal cistemae (TC) of the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. PtdIns and PtdIns4P have been detected in both membrane systems whereas PtdIns(4,5)P, (290 pol/mol phospholipid) is confined only to 'FT. A much greater pool of PtdIns(4,5)P, seems, however, to be located in the sarcolemma away from the triadic junction.Key wor&: Phosphoinositide; PtdIns(4,5)P,; Phospholipase C, Inositol 1,4,56sphosphate;Triad; Skeletal muscle IntruduetIonThe concept that inositol 1,4,5-triphosphate (Ins-(1,4,5)PJ acts as an intracellular chemical transmitter in the fast excitation-contraction coupling process of skeletal muscle has been put forward by several groups [l-3]. There are, however, severe objections to this idea which have been worked out in detail by [4]. A second concept favours a messenger action of Ins(1,4,5)P, on a larger time scale aiming at the excitation-contraction coupling mechanism and/or the energy metabolism of the skeletal muscle fibre [5]. A key event of the Ins( 1,4,5)P,-signalling pathway is the cleavage of Ins(1,4,5)P, from phosphatidylinositol4,5-biphosphate (PtdIns(4,5)Pb by phospholipase C (PLC) [6] that has to fit in quite different time frames depending on which of the concepts is realised in skeletal muscle. It is well established that the activity of skeletal muscle PLC is strongly regulated by myoplasmic Ca2+ in a concentration range that is reached during muscle activation [7,8]. Up to now it is unknown whether this Ca2'-sensitivity, which is common to all known PLC isoforms [9], represents the primary event for PLC-activation or whether a different membrane borne process switches on the enzyme Ca2+-independently. A putative assignment of the activation mechanism to one of the established classes (G protein activated for PLC-B; receptor tyrosine kinase activated for PLC-y [6,9]) is not possible because a PLC isoform of skeletal muscle has not yet been identitied by protein or cDNA sequencing. There is, however, evidence that two PLC, i.e. a cytosolic and a membrane associated form, coexist in skeletal muscle [7,8] and that the myoplasmic enzyme might be different from the known PLC families [lo]. The membrane associated enzyme is localised mainly at the TT [7,8]. Membranes of the SR seem either to be devoid of this enzyme [7] or contain marginal PLC activity [8]. Compared to the knowledge accumulated on skeletal muscle PLC only little is known about membrane localisation and content of its substrate, PtdIns(4,5)P,. In the present work we have studied in detail the total concentration and localisation of phosphatidylinositol (Ptdlns), phosphatidylinositol4-phosphate (PtdI&P), and PtdIns-(4,5)P, in TT membranes and in membran...

SUMMARY1. The contractile apparatus of mechanically skinned muscle fibres of frog can be reversibly activated by Ba2+ and Cd2 .2. The maximum force induced by both Ba2+ and Cd2+ is the same as that induced by Ca2+ and Sr2+.3. The ionic concentrations of the divalent cations required to induce 50 % of the maximum activated force at 1 mM-Mg2+, pH 7410, 22 TC and 250 mm ionic strength are about 8 x 10-7 M for Ca2+, 5 X 10-6 M for Cd2+, 2-6 x 10-5 M for Sr2+ and 7 x 10-4 M for Ba2+.4. Exposure of the skinned fibre to relatively low Ni2+ concentrations (between 10-6 and 10-5 M) resulted in a transient force response accompanied by an irreversible change in the ability of the preparation to develop force.5. The Ba2+-and Cd2+-activation curves are considerably flatter than the corresponding curves for Ca2+ and Sr2+.6. An increase in Mg2+ concentration from 1 to 3 mm decreased the sensitivity of the contractile apparatus to Ba2+ by a factor of about 1P5 without affecting the maximum force response.7. The Ca2+-activation curve was modified in the presence of subthreshold concentrations of Ba2+ and the results indicate that Ba2+ could have both an activating and an inhibitory action on the Ca2+-activated force.8. A kinetic model which can quantitatively explain the results for activation of contraction by Ba2+ and Ca2+, is described.