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- supplemental material - Supplement 1. Combined [Ca2+]i and [Ca2+]ER recordings from DRG neurons. Cytosolic and ER luminal Ca2+ concentrations were simultaneously monitored using fura-2 (Kd ≈ 0.22 μM) and Mag-fluo-4 (Kd ≈ 22 μM), respectively. Cells were initially loaded with Mag-fluo-4/AM at a concentration of 7.5 μM in 0.02 % Pluronic F-127 at 37oC for 45 min and then washed 3 times in dye-free HEPES-buffered Hank�s solution (HHSS; see Materials and Methods); cells were further loaded with fura-2/AM (5 μM; 30 min at 22oC in 0.02 % Pluronic F-127), followed by a 30 min wash in dye-free HHSS buffer. Cells were imaged with a CCD-camera based microfluorimetry system (Till Photonics, Germany) mounted on an inverted microscope (Olympus IX-71) equipped with a 40x (oil immersion; NA=1.35) objective. Fura-2 and Mag-fluo-4 were alternately excited at 340/380 and 475 nm, respectively, and fluorescence measured at 525 (40) nm. Images were collected at each of the excitation wavelengths every 2 s (A) or every 5 s (B and C). The fluorescence ratio (R=F340/F380) of fura-2 was converted to [Ca2+]i according to the formula: [Ca2+]i = Kdβ(R-Rmin)/(Rmax-R) (Grynkiewicz et al., 1985). The dissociation constant (Kd) used for fura-2 was 224 nM. Ionomycin was used to determine the calibration constants, as described in Materials and Methods: Rmin=0.17, Rmax=2.71, β=5.6. Changes in [Ca2+]ER were expressed as ΔF/F=(F-Fo)/Fo, where F was the current Mag-fluo-4 fluorescence value and Fo was the fluorescence value before treatments. Fluorescence was always corrected for background. A, Caffeine (10 mM) induced Ca2+ release from ryanodine-sensitive stores. This led to a rapid rise in [Ca2+]i and a complementary decrease in [Ca2+]ER. After reaching its peak, [Ca2+]i began to recover despite the presence of caffeine and continuous Ca2+ flux from the stores. This is likely explained by the competition between Ca2+ release and Ca2+ clearance processes. As the stores deplete, the rate of Ca2+ release subsides; the Ca2+ clearance mechanisms turned on by the [Ca2+]i elevation begin to dominate leading to the recovery of [Ca2+]i. Indeed, the time course of [Ca2+]i changes correlated with the rate of Ca2+ mobilization from the stores, d[Ca2+]ER/dt (blue). A portion of the d[Ca2+]ER/dt trace during the refilling phase was truncated for clarity. B, A representative combined recording of [Ca2+]i (black) and [Ca2+]ER (green) during the standard refilling protocol used in the study. The stores were depleted by 5 μM CPA in Ca2+-free media. The maximal rate of CPA-induced Ca2+ mobilization from the stores was 15-20% of that induced by caffeine. The refilling process was initiated by switching to control HHSS buffer containing 1.3 mM Ca2+. The rate of refilling during the first and the second trial was highly reproducible: k1=0.29�0.03 min-1 and k2=0.28�0.03 min-1 (n=12). The d[Ca2+]ER/dt trace (blue) has been truncated during the refilling phase for clarity. C, CPA (5 μM) blocked the refilling phase, consistent with Ca2+ reuptake via SERCA. The same paired refilling protocol used in B was used here, except CPA remained in the solution during the second trial. The recording is representative of 5 experiments. Addition of extracellular Ca2+ during the second trial induced a rapid elevation of [Ca2+]i, whereas the stores remained depleted in the presence of CPA. [Ca2+]ER completely recovered after CPA was washed out. The recovery phase was slowed relative to the control refilling process because of the relatively slow (5-10 min) wash out of CPA. Note that during the control refilling protocol, CPA was washed from the bath for 10 min prior to initiating refilling. The [Ca2+]i overshoot above baseline likely resulted from capacitative Ca2+ influx (Usachev and Thayer, 1999) because the stores remained depleted in the presence of CPA. References Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450. Usachev YM, Thayer SA (1999) Ca2+ influx in resting rat sensory neurones that regulates and is regulated by ryanodine-sensitive Ca2+ stores. J Physiol 519:115-130.