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Ca2+ release from the sarcoplasmic reticulum (SR) by the IP3 receptors (IP3Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP3R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca2+ release from the IP3R in smooth muscle. The presence of FKBP12 was confirmed in colonic myocytes and co-immunoprecipitated with the IP3R. In aortic smooth muscle, however, although present, FKBP12 did not co-immunoprecipitate with IP3R. In voltage-clamped single colonic myocytes rapamycin, which together with FKBP12 inhibits mTOR (but not calcineurin), decreased the rise in cytosolic Ca2+ concentration ([Ca2+]c) evoked by IP3R activation (by photolysis of caged IP3), without decreasing the SR luminal Ca2+ concentration ([Ca2+]l) as did the mTOR inhibitors RAD001 and LY294002. However, FK506, which with FKBP12 inhibits calcineurin (but not mTOR), potentiated the IP3-evoked [Ca2+]c increase. This potentiation was due to the inhibition of calcineurin; it was mimicked by the phosphatase inhibitors cypermethrin and okadaic acid. The latter two inhibitors also prevented the FK506-evoked increase as did a calcineurin inhibitory peptide (CiP). In aortic smooth muscle, where FKBP12 was not associated with IP3R, the IP3-mediated Ca2+ release was unaffected by FK506 or rapamycin. Together, these results suggest that FKBP12 has little direct effect on IP3-mediated Ca2+ release, even though it is associated with IP3R in colonic myocytes. However, FKBP12 might indirectly modulate Ca2+ release through two effector proteins: (1) mTOR, which potentiates and (2) calcineurin, which inhibits Ca2+ release from IP3R in smooth muscle

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To study the contribution of the Na+‐Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]c were evoked by either depolarisation (−70 mV to 0 mV) or by release from the SR by caffeine (10 mm) or flash photolysis of caged InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+‐induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation‐evoked rise in [Ca2+]c. However, although Ca2+ was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation‐evoked Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+‐Ca2+ exchanger activity. Thus inhibition of the Na+‐Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+‐Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+‐Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+‐Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.

Sarcolemma Ca2+ influx, necessary for store refilling, was well maintained, over a wide range (‐70 to + 40 mV) of membrane voltages, in guinea‐pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP3‐evoked Ca2+ transients. This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca2+ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. ‐70 mV), Ca2+ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage‐gated Ca2+ channels were largely responsible. Sarcolemma voltage‐gated Ca2+ currents were not activated following store depletion. Removal of external Ca2+ or the addition of the Ca2+ channel blocker nimodipine (1 μM) inhibited store refilling, as assessed by the magnitude of InsP3‐evoked Ca2+ transients, with little or no change in bulk average cytoplasmic Ca2+ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca2+ gradient. Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca2+ gradient. Store access to the gradient allows InsP3‐evoked Ca2+ signalling to be maintained over a wide voltage range in colonic smooth muscle.