• Muscle Contraction C1-C6 PC1-PC11

University of Heidelberg (2006) Proc Physiol Soc 4, C6

Oral Communications: Bradley S. Launikonis1, Eduardo Ríos1

1. Physiology, Rush University, Chicago, IL, USA.

In most eukaryotic cells, depletion of the internal Ca2+ store activates store-operated calcium entry (SOCE). We used shifted excitation and emission ratioing of fluorescence (Launikonis et al. 2005) to image mag-indo-1 trapped in the t-system of mechanically skinned rat skeletal muscle fibres, simultaneously with rhod-2 in the cytoplasm, to measure for the first time SOCE during intracellular Ca2+ release. Ca2+ release was induced pharmacologically, to avoid activation of voltage-dependent contributions to Ca2+ flux. Spatially and temporally resolved images of t-system [Ca2+]([Ca2+]t-sys) allowed estimation of Ca2+ flux from the rate of change of [Ca2+]t-sys. An initial uptake of Ca2+ by the t-system was observed during the release of Ca2+ from sarcoplasmic reticulum (SR) at rates approaching 1 mM s-1 (relative to t-system volume; n = 8 fibres), depending upon cytoplasmic buffering conditions. Activation of SOCE occurred prior to the peak of the cytoplasmic Ca2+ transient, as indicated by an abrupt decay in [Ca2+]t-sys. Cytoplasmic Ca2+-buffering conditions did not affect SOCE activation or Ca2+ entry through the t-system membrane. Once SOCE was activated, Ca2+ entry flux was exponentially dependent (rate constant, 0.9 s-1) on the concentration gradient, essentially [Ca2+]t-sys, as well as the transmembrane potential (n = 16 fibres). [Ca2+]t-sys could only recover upon a net uptake of Ca2+ by the SR in a low [Mg2+]cyto solution (n = 4 fibres; see [Ca2+]SR measurements under similar conditions in Launikonis et al. 2006), indicating SOCE termination required SR refilling. Depletion without recovery of [Ca2+]SR in caffeine allowed activation but not termination of SOCE (n = 12 fibres). The concomitant change in sign of the t-system Ca2+ flux with depletion and recovery of [Ca2+]SR is diagnostic of SOCE and validates the measurements. Ca2+ entry flux was 13.3 μM s-1 during SOCE in the polarized cell at physiological [Ca2+]t-sys. This flux is consistent with SR refilling rates mediated by SOCE in experiments with intact EDL fibres of rat (Kurebayashi & Ogawa, 2001). The experiments also showed that activation of SOCE was regulated by a local signal, did not require full depletion and was not clearly graded with depletion. Termination of SOCE required a net increase in [Ca2+]SR.

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• Launikonis, BS
• Ríos, E