Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.

The mammalian rod synapse transmits a binary signal (one photon or none) using tonic, rapid exocytosis. We constructed a quantitative, physical model of the synapse. Presynaptically, a single, linear active zone provides docking sites for approximately 130 vesicles, and a "ribbon" anchored to the active zone provides a depot for approximately 640 vesicles. Postsynaptically, 4 processes invaginate the terminal: 2 (known to have low affinity glutamate receptors) lie near the active zone (16 nm), and 2 (known to have high affinity glutamate receptors) lie at a distance (130-640 nm). The presynaptic structure seems designed to minimize fluctuations in tonic rate owing to empty docking sites, whereas the postsynaptic geometry may permit 1 vesicle to evoke an all-or-none response at all 4 postsynaptic processes.

We have characterized the ␣-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of ␣7-containing receptors that bind ␣-Bgt with high affinity (K d ϭ 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and ␣-Bgt block ϳ90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric ␣7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for ␣7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and ␣7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and ␣7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (>3.8 ,um) and bathed in a 'high-Ca2>' Ringer ([Ca2] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (±0.01, SEM) X 10-6 cm2 S-1 (N = 5; 16CC), it is estimated that-85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3to 4-fold, the indicator's apparent dissociation constant for Ca 2 (KD), which is estimated to be 1.1-1.6 MM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate f, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, f4 was estimated to be 0.15 (±0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 MM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length ('2.7 ,um), and containing a nonperturbing concentration of indicator (<0.2 mM), [Ca2+], is estimated to be 0.18-0.27 uM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RM}x (the values of R that would be measured if all indicator molecules were in the Ca2+-free and Ca2+-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+], from R are analyzed and discussed.

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca 2+ , although the absolute magnitude of the Ca 2+ current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca 2+ to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca 2+ was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca 2+ -sensing dye Fluo-4, and found that acidification increased [Ca 2+ ]i in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca 2+ , our single cell data strongly challenges the view that Ca 2+ entry through the ASIC1a receptor itself contributes to this response.

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.