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1 Departamento de Biofísica, Facultad de Medicina, Universidad de la República, Gral Flores 2125, Montevideo, Uruguay Email: gpizarro{at}fmed.edu.uy
The field of intracellular [Ca2+] measurements in skeletal muscle started in 1966 with the pioneering work of Jobsis & O'Connor (1966) using the metallochromic indicator murexide. A derivative of murexide, tetramethyl murexide (TMX), was introduced about 10 years later, mostly to measure intravesicular Ca2+ in fragmented SR. Its use in cellular studies was hindered by the fact that TMX passes through membranes including that of the sarcoplasmic reticulum (SR). Maylie et al. (1987) reported that when Ca2+ release was triggered by an action potential TMX gave a biphasic signal, with an early component that originated in the myoplasm and a late sustained component of opposite polarity probably coming from the dye in the SR lumen. In those days the focus was on the myoplasmic Ca2+ transient, and therefore the signal coming from the SR was mostly a nuisance and TMX fell out of favour despite its simple stoichiometry, fast response and low binding to cytoplasmic constituents. After many generations of fancy fluorescent dyes and a wealth of experimentation some workers in the field turned their attention to the inner workings of the SR. Only recently the time course and amount of Ca2+ released from the sarcoplasmic reticulum Ca2+ has been directly measured in living skeletal muscle cells (Kabbara & Allen, 2001; Rudolf et al. 2006; Launikonis et al. 2006). In this issue of The Journal of Physiology, Pape et al. (2007) add to these direct measurements with the first study using cut fibres under voltage clamp.
For that they put the permeation properties of TMX to good use. In the presence of 20 mM EGTA in the myoplasm (an experimental condition known to strongly buffer the free Ca2+ transient but not fundamentally modify Ca2+ release), they abolished the myoplasmic component isolating the depletion signal that originated in the SR. The authors combined the TMX measurement with phenol red (PhR) added to the myoplasm to track the protons displaced from EGTA upon Ca binding. The TMX signal reports the time course of the free Ca2+ in the SR lumen while the PhR signal follows the time course of the total, free plus bound, Ca in the SR.
With these tools at hand, the saturation curve of calsequestrin (Csq) ‘in situ’ was constructed. Csq showed high capacity and bound Ca2+ cooperatively with a Hill coefficient close to 3. Based on this analysis they concluded that the resting free Ca2+ in the SR was in the range of the dissociation constant of Csq. Their best estimate of intra-SR resting free Ca2+ of 1.22 mM is generally consistent with in vitro determinations of the dissociation constant of Csq. The time course of the two signals superimposed for SR content up to one half of resting level, consistent with strong buffering in this range. Below 50% of the resting content, as the total Ca2+ in the SR approached zero, the TMX signal exceeded the PhR signal.
The emerging picture from this work is a relatively simple one. Csq is the main source of Ca2+ for a single twitch (which releases to the myoplasm less than 12% of the SR resting content) and does not appear to play a regulatory role for the release mechanism. This latter finding is at variance with the important regulatory function of Csq in heart cells and with previous reports in fragmented SR and permeabilized fibres from skeletal muscle. Pape et al. (2007) failed to observe an increase in intra-SR free Ca2+ preceding Ca2+ release, a phenomenon reported both by Ikemoto et al. (1991) and Launikonis et al. (2006) under submaximal stimulatory conditions. Whether this is due to differences in the stimuli (voltage versus pharmacologically triggered release), difference in spatial resolution of the measurements or differences in other experimental conditions remains an open question.
Another important difference with other recent intra-SR measurements is a kinetic one. Pape et al. (2007) found a delay of 7 ms between the free Ca2+ signal and the total Ca signal. They found this to be consistent with diffusional delays in the longitudinal SR from which most of their TMX signal originates. This delay observed with TMX seems substantially less than that reported by Launikonis et al. (2006) in confocal studies using mag-indo-1. Pape et al. (2007) argue that the longer delay is not likely to arise from the same mechanism. Whether the difference originates in the properties of the indicators or experimental conditions should be further explored. In any case the availability of various technical approaches and the interesting discrepancies pointed above anticipate a promising future of intense experimental studies of intra-SR Ca2+.
References
Ikemoto N, Antoniu B, Kang JJ, Meszaros LG & Ronjat M (1991). Biochemistry 30, 5230–5237.[CrossRef][Medline]
Jobsis FF & O'Connor MJ (1966). Biochem Biophys Res Commun 25, 246–252.[CrossRef][Medline]
Kabbara AA & Allen DG (2001). J Physiol 534, 87–97.
Launikonis BS, Zhou J, Royer L, Shannon TR, Brum G & Rios E (2006). Proc Natl Acad Sci U S A 103, 2982–2987.
Maylie J, Irving M, Sizto NL, Boyarsky G & Chandler WK (1987). J General Physiol 89, 145–176.
Pape PC, Fénelon K, Lamboley CRH & Stachura D (2007). Physiol 581, 323–371.
Rudolf R, Magalhaes PJ & Pozzan T (2006). J Cell Biol 173, 187–193.
Related Article
J. Physiol. 2007 581: 319-367.
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