In non-excitable cells, the emptying of inositol 1, 4, 5-trisphosphate (InsP₃)-sensitive Ca²⁺ stores activates Ca²⁺ entry through plasma membrane channels, a process know as store-operated or capacitative calcium entry. The most widely distributed and best characterized store-operated Ca²⁺ current is the Ca²⁺ release activated Ca²⁺ current (I_CRAC). I_CRAC can be measured directly using the whole-cell patch-clamp technique. However, I_CRAC is usually recorded using very high concentrations (several mM) of Ca²⁺ chelators in the recording pipette. Under more physiological conditions of weak buffering, InsP₃ generally fails to activate any detectable I_CRAC unless sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps are blocked with thapsigargin.

Increased Ca²⁺ uptake by mitochondria prevents SERCA-mediated refilling of stores and enables InsP₃ to consistently activate I_CRAC in weak buffer. We have now explored the effects of mitochondrial Ca²⁺ buffering on the ability of receptor stimulation to elicit I_CRAC in weak buffer. Whole-cell patch-clamp experiments were carried out on rat basophilic leukaemia (RBL-1) cells. I_CRAC was monitored by applying voltage ramps (-100 to 100 mV in 50 ms) at room temperature (20-25 °C) from a holding potential of 0 mV. Data are presented as means ± S.E.M. and statistical analysis was carried out using either Student’s t or non-parametric Mann-Whitney tests.

Stimulation of muscarinic receptors, which engage the phosphoinositide pathway, with carbachol generally failed to activate any detectable I_CRAC under standard conditions (-0.30 ± 0.08 pA pF^-1; n = 5), even when the pipette composition was systematically altered (Cs⁺ vs. K⁺, Cl^– vs. Glu^–, ± ATP). However, when a cocktail (malate, pyruvate, NaH₂PO₄, cAMP and GTP) that is known to be important for maintaining functional respiring mitochondria (Gunter & Pfeiffer, 1990) was included in the pipette, carbachol evoked a larger I_CRAC (-1.23 ± 0.15 pA pF^-1; n = 5, P < 0.0005 vs. control using Student’s unpaired t test). The effects of the cocktail were less prominent following reduced mitochondrial Ca²⁺ uptake.

Our findings suggest the ability of an agonist to activate I_CRAC seems to be determined, at least in part, by the state of mitochondria. Mitochondria can compete effectively with SERCA pumps such that the stores deplete sufficiently for macroscopic I_CRAC to activate.

J.A.G. is supported by a Marie Curie EU Posdoctoral Fellowship.