Accumulation of inorganic phosphate (P i) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P i into SR are fully established. r We showed that P i promoted Ca 2+ release at concentrations below 10 mM and decreased it at higher concentrations. This decrease correlated well with that of [Ca 2+ ] SR. r Pre-treatment of permeabilized myofibres with 2 mM Cl − channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of P i. r The biphasic dependence of Ca 2+ release on [P i ] is explained by a direct effect of P i acting on the SR Ca 2+ release channel, combined with the intra-SR precipitation of Ca 2+ salts. The effects of 9AC demonstrate that P i enters the SR via a Cl − pathway of an as-yet-undefined molecular nature.

In excitation–contraction coupling, voltage-sensing modules (VSMs) of CaV1.1 Ca2+ channels simultaneously gate the associated pore and Ca2+ release channels in the sarcoplasmic reticulum. Ferreira Gregorio et al. find that VSMs adopt two inactivated states, and the degree of inactivation is dependent on external Ca2+ and the mouse strain used.

This study describes the development and characterisation of software to enable automatic detection and analysis of Ca 2þ sparks within x-y image stacks, implemented as a plugin within the open source image analysis platform, ImageJ. The aim was to implement a "conventional" algorithm whereby sparks were identified by applying a threshold (q) to the normalised (F/F 0 ) image: q = background fluorescence within the cell þ SD * 'ε', a user defined variable. A 2 stage interactive method with a graphical user interface (GUI) was used to ensure precise identification of the cell boundary and creation of a binary cell mask, which is subsequently used to exclude all regions outside the cell. The algorithm separates spark detection and analysis, allowing image processing to be applied independently at both stages. Filters also allow exclusion of events based on spark width or morphology. Novel methods are included to allow correction of time dependent changes in background fluorescence (e.g. due to bleaching), which would otherwise compromise spark detection by thresholding. The main outputs (amplitude, width, duration and spark mass) are presented in tabular form. In addition, an interactive GUI allows each spark to be examined, along with its measurements, and the associated Gaussian curve fit. A "Kill" button allows obvious errors in detection to be excluded. The performance of the algorithm was tested both on synthesised images (values of ε ranging from 3.0-4.2 and signal to noise ratios of 2, 3 or 4) and on x-y confocal fluorescence images from fluo-3 loaded rat ventricular myocytes. In both cases the performance was comparable to that reported previously for threshold based detection methods applied to line-scan images.

The mutant hKv1.3_V388C channel shows inward current at potentials more negative than 100 mV when the central apore is closed (Pruetting et al., JBC 286:20031-20042). We believe that the V388C mutation in hKv1.3 channels opens a new pathway (s-pore) behind the K þ conducting apore. The entry of the spore from the extracellular side is presumably located at the backside of Y395 (Shaker position 445) and F384 (Shaker position 434). One spore seems to be formed between two neighboring S6 subunits and it runs parallel to the apore. For a more detailed characterization of the spore, we created a concatamer consisting of three hKv1.3_V388C asubunits linked together. Overexpression of this trimeric hKv1.3_V388C channel in COS7 cells yielded typical spore currents at potentials more negative than 100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channels were similar: currents at potentials more negative than 100 mV were not carried by protons or chloride ions and could not be reduced by peptide toxins (CTX, MTX) or TEA. The spore was mostly permeable to Na þ and Li þ . For the determination of the path of the spore in the hKv1.3_V388C channel we mutated single amino acids in the hKv1.3_V388C mutant channel that maybe involved in spore formation. Two of these mutant channels (hKv1.3_V388C_W384F, hKv1.3_V388C_Y395W) reduced current through the spore indicating that these positions might line the spore.