Inhibitors of calmodulin-dependent phosphorylation simultaneously inhibit calcium uptake and calcium-dependent ATPase activity in skeletal muscle sarcoplasmic reticulum and transiently induce calcium release

Under adequate experimental conditions calmodulin antagonists like compound 48/80 do not dissociate calcium uptake from the calcium-dependent ATP hydrolysis of skeletal muscle sarcoplasmic reticulum membranes but simultaneously inhibit both processes. Apart from the agent's pump inhibiting effect, they interact with the caffeine sensitive calcium channel in the sarcoplasmic reticulum causing a rapid transient calcium release

Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel

Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li+ < NH+ 4 < K−∼ Cs+≤ Na+) and anions (gluconate− < Cl− < NO3 −∼ ClO4 −∼ SCN−) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25–11 m) and different degrees of cooperativity (n= 1.5–4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m −1· sec−1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M−1· sec−1). The nitrogen bases, NH+ 4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine–protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1–3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine

Arrangement of Proteins and Lipids in the Sarcoplasmic Membrane

The number of amino residues present in the proteins of the sarcoplasmic reticulum which can react with Fluram has been determined in native and sonicated SR vesicles. Sonication increases the number of amino groups accessible to Fluram from 0.57 to 0 .8 7 μmol·mg prot.-1. This increase indicates that 66% of the amino residues are present in the external and 34% in the internal membrane leaflet. The distribution of the amino phospholipids is computed from the distribution of Fluram in the membrane proteins in con­junction with the relative distribution of Fluram between protein and lipid in native and sonicated vesicles. The distribution of the calcium transport protein has been approximated under different assumptions concerning the distribution of the residual protein and taking into account that 15% of the membranes of the SR vesicles might have changed their sideness during preparation

Modulation by monovalent anions of calcium and caffeine induced calcium release from heavy sarcoplasmic reticulum vesicles

Both calcium and caffeine induced calcium release from actively loaded heavy sarcoplasmic reticulum vesicles were studied to analyze the dependence of both activities on the composition of the release medium with respect to monovalent anions. Calcium is unable to induce net calcium release while caffeine remains effective as releasing agent when the experimental media contain neither chloride nor nitrate ions. Caffeine induced calcium release is not suppressed by chelating residual medium calcium (approximately 0.5-1 microM) with 2 mM EGTA added 15 s prior to 10 mM caffeine. Calcium release from vesicles loaded in media containing 0.2 M gluconate as monovalent anion is induced when the medium is supplemented with chloride or nitrate. The release amplitude increases linearly when K-gluconate is replaced by KCl. At constant ionic strength the release amplitude becomes maximal at a chloride concentration of 0.2 M. The chloride effect completely disappears when 2 mM EGTA are added simultaneously. When chloride is replaced by nitrate, as releasing agent, maximal release is achieved already by addition of 0.1 M K-nitrate. The releasing effect of nitrate can only partially be suppressed by EGTA. The different effectiveness of gluconate, chloride and nitrate as calcium release supporting ions corresponds to their activating effect on the binding of ryanodine to the calcium release channel in the vesicular membranes

Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high-affinity ryanodine binding

The occupancy of high-affinity ryanodine-binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP-dependent calcium accumulation and caffeine-induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine-induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine-induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drug's effectiveness in living muscles

The Inhibition of the Calcium Transport ATPase of the Sarcoplasmic Reticulum by Fluorescamine: Evidence for an Oligomeric Functional Unit of the Calcium Transport System

The labeling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluorescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphorylation by [gamma-32P]ATP and [32P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase, it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules

Calcium Binding to Sarcoplasmic Membranes

ATP-Supported calcium accumulation of sarcoplasmic vesicles is abolished when the vesicles are disrupted or made leaky. The native as well as the leaky sarcoplasmic membranes are characterized by a high intrinsic affinity for calcium which does not depend on ATP. Neither the number of the calcium binding sites nor their affinity for calcium is affected when the membranes are deprived of the phospholipids which are essential for the activity of the transport ATPase

Invariance of Stoichiometry of the Sarcoplasmic Reticulum Calcium Pump at Physiological Calcium Concentrations – a Reevaluation

The decline of the transport ratio of the sarcoplasmic calcium pump observed in a recent study (A. results from the retardation of calcium oxalate precipitation at low calcium/protein ratios. The prevailing high internal calcium level supports a rapid calcium backflux and a compensatory ATP hydrolysis during net calcium uptake which reduces the transport ratio. Yet, the determined calcium back­ flux does not fully account for the decline of the transport ratio. A supposed modulation of the stoichiometry of the pump by external calcium (0.1 μм) is at variance with results of previous studies showing a constant transport ratio of two in the same calcium concentration range

Calcium gradient dependent pyrophosphate formation by sarcoplasmic vesicles

Pyrophosphate Synthesis, Sarcoplasmic Reticulum, Calcium Accumulation The vesicles of the sarcoplasmic membranes synthesize pyrophosphate from inorganic phosphate. Pyrophosphate synthesis proceeds as long as a calcium gradient is maintained across the vesicular membranes. Pyrophosphate synthesis is inhibited by low concentrations of nucleoside triphosphates