Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres

Abstract

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ( Calsequestrin is a high-capacity Ca binding protein located in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal muscle (MacLennan & Wong, 1971). One function proposed for calsequestrin is that it rapidly buffers [Ca 2+ ] in the sarcoplasmic reticulum (denoted [Ca 2+ ] SR ). In this way, local depletion of Ca 2+ would be avoided and the driving force for Ca 2+ release maintained. In support of this idea, the in vitro kinetics of the Ca-calsequestrin binding reaction appear to be fast ('rates of calcium association and dissociation are faster than 1 ms −1 ; Prieto et al. 1994) indicating that they would not be rate limiting for physiological SR Ca 2+ release. Therefore, unless its kinetic properties change significantly in intact muscle, calsequestrin should essentially be in instantaneous equilibrium with [Ca 2+ ] SR during physiological stimulation. The ability of calsequestrin to act as a local buffer should then depend on the amount of calsequestrin, its steady-state binding curve with Ca, and the location on this curve determined by [Ca 2+ ] SR . Essentially all of the information on calsequestrin's binding properties thus far has come from studies with purified calsequestrin or with disrupted preparations, principally terminal cisternae vesicles. The main aim of this article is to evaluate these properties under more physiological conditions during depolarization-induced Ca 2+ release.The main experiments in this article involved measurements of two optical signals in response to voltage-clamp stimulation, one signal approximately proportional to total SR Ca content and the other driven by changes in [Ca 2+ ] SR . The concentration of total SR Ca content (denoted [Ca T ] SR ) were obtained from absorbance measurements of phenol red (the first optical signal) with the EGTA-phenol red method . [Ca 2+ ] SR was monitored with the absorbance indicator-dye tetramethylmurexide (TMX, the second optical signal), an application suggested by the results of Maylie et al. (1987). They described TMX-related absorbance changes ( A TMX ) from cut muscle fibres equilibrated with a low concentration of EGTA (0.1 mm) in response to action-potential stimulation. The A TMX signal had two components, an early transient component consistent with an increase in [Ca 2+ ] in the myoplasm followed by a rapid decrease to a somewhat maintained signal of opposite sign to the initial transient component. They proposed that the maintained signal was due to a decrease in [Ca 2+ ] SR . In a subsequent study, measurements were carried out using modified Ca 2+ indicators which were essentially TMX or a similar dye, murexide, with two charged groups added to prevent their entry into the SR (Hirota et al. 1989). As predicted, the action-potential-generated, dye-related A signals showed a rapid transient component that ...

Calcium indicators and calcium signalling in skeletal muscle fibres during excitation–contraction coupling

“…In contrast, use of TMX, the other purpurate indicator in Table 1, is problematic because TMX has a significant membrane permeability (Ogawa et al, 1980; Ohnishi, 1979). In both cut and intact fibres, the ΔA signal from TMX is biphasic, with an early component that monitors Δ[Ca 2+ ] and a later component (of opposite sign) that appears to reflect a decrease in [Ca 2+ ] within the SR lumen (Maylie et al, 1987a; Pape et al, 2007). Because the time courses of the two TMX components overlap, estimation of the Δ[Ca 2+ ] waveform from the ΔA of TMX is problematic.…”

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

“…Given that Casq1 provides much of the calcium that signals muscle contraction (25,26), the buffering features found in solution should transfer to the storage properties of the SR in cells. Indeed, strongly cooperative calcium buffering by the SR was first documented by Pape et al (31), who showed with frog muscle that the calcium buffering power of the empty SR is negligible compared with the value at rest, a property they attributed to calsequestrin. Working on mouse muscle, Royer et al (19) confirmed the attribution, quantified the contribution by Casq1 at 75% of released calcium (26), and equated it to the hump of calcium flux caused by a large depolarization (19).…”

Comparison of sarcoplasmic reticulum calcium content in atrial and ventricular myocytes of three fish species

“…In particular, experiments on multicellular cardiac preparations strongly suggest that contribution of SR Ca 2ϩ release to e-c coupling of the fish heart is significantly less than SL Ca 2ϩ entry, because in the majority of fish species examined, the force of cardiac contraction is weakly responsive to Ry. At physiological heart rates and temperatures, Ry inhibition of cardiac contraction varies from 0 to 32% (2,12,14,33,49), the most notable exceptions being tuna and an eurythermic neotropical teleost, Synbranchus marmarotus (28, 37), species in which Ry inhibition of force generation ranges from 30 to 53%.On the other hand, it has been found that in cardiac myocytes of rainbow trout, caffeine-releasable Ca 2ϩ stores are massive, almost an order of magnitude larger than in mammalian cardiac myocytes and approaching to the values of the mammalian skeletal muscle fibers (26,35,45). Thus, there is a striking mismatch between massive SR Ca 2ϩ stores and relatively weak effect of Ry on cardiac contraction at least for the trout heart (2,22,33,46), which still lacks a mechanistic explanation and raises several questions.…”

“…On the other hand, it has been found that in cardiac myocytes of rainbow trout, caffeine-releasable Ca 2ϩ stores are massive, almost an order of magnitude larger than in mammalian cardiac myocytes and approaching to the values of the mammalian skeletal muscle fibers (26,35,45). Thus, there is a striking mismatch between massive SR Ca 2ϩ stores and relatively weak effect of Ry on cardiac contraction at least for the trout heart (2,22,33,46), which still lacks a mechanistic explanation and raises several questions.…”