Titration of synaptotagmin I expression differentially regulates release of norepinephrine and neuropeptide Y.

Synaptotagmin (syt) I is a Ca(2+) sensor that has been thought to trigger all vesicle secretion with similar mechanisms. However, given the calcium and stimulation requirements of small clear, and large dense core vesicles, we hypothesized that syt I expression differentially regulates vesicle release. Therefore, in this study, we generated multiple stable cell lines of PC12 cells that each had a different and stable level of syt I expression. We determined the functional effects of titrated syt I expression on transmitter release from the two vesicle types, and showed that the transmitters, norepinephrine (NE) and neuropeptide Y (NPY), each have a threshold level of syt I expression required for their release that is different for the two transmitter types. We used carbon fiber amperometry to measure release of NE from single vesicles, and found that release ranged from 50% to 100% in the syt I-targeted cells compared to release from control cells. We used an immunoassay to measure NPY release and found that NPY release was abolished in cells that had abolished syt I expression, but cell lines that expressed 50-60% of control levels of syt I exhibited NPY release levels comparable to release of NPY from control cells. Furthermore, the vesicle fusion pore exhibited a reduced open duration when syt I was abolished, but a longer open duration time for 50% syt I expression than control cells. Therefore, vesicles have a threshold for syt I that is required to control opening of the fusion pore, expansion, and full fusion to release large dense core proteins, but not for full fusion of the small molecules like NE.

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.

Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells.

Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress Ca(2+) channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2 suggesting the involvement of P(2Y) purinergic receptors. Prepulses to positive potentials that relieve the Ca(2+) channel block largely relieve the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca(2+) influx there is no apparent change in the relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes secretion by inhibiting Ca(2+) influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca(2+) channels, may be an important regulator of catecholamine release in chromaffin cells.

Evidence of elevated intracellular calcium levels in weaver homozygote mice.

1. A mutation in the G-protein-linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2. We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild-type neurons. 3. High-K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild-type neurons. 4. weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX-314. 5. QX-314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6. These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

Activation of nicotinic acetylcholine receptors augments calcium channel-mediated exocytosis in rat pheochromocytoma (PC12) cells.

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca2+-permeable nAChRs or with voltage steps to activate voltage-dependent Ca2+ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at -80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd2+ to block voltage-activated Ca2+ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca2+ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca2+ channels alone. Our data suggest that Ca2+ influx via nAChRs affects Ca2+-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca2+ entry pathway underlying Ca2+-dependent cellular processes such as exocytosis.

Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors.

We have characterized the alpha-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of alpha7-containing receptors that bind alpha-Bgt with high affinity (Kd = 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and alpha-Bgt block approximately 90% 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 alpha7/5HT3 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 alpha7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and alpha7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and alpha7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Mammalian rod terminal: architecture of a binary synapse.

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.

Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.

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.

Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.

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 (approximately 3.8 microns) 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; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, 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, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. 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 RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2.

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.

Trimethyltin alters membrane properties of CA1 hippocampal neurons.

Trimethyltin produces pathological changes in the hippocampus, but the physiological mechanisms underlying its toxicity remain unclear. Intracellular recordings of CA1 neurons in rat hippocampal slices were conducted during bath application of 50 and 100 microM trimethyltin and 50 and 200 microM dimethyltin. Trimethyltin slowly depolarized the membrane potential without spontaneous spiking, and decreased input resistance and time constant. Trimethyltin decreased, in a dose-dependent manner, both the elicited action potential and the orthodromically-stimulated action potential amplitudes; increased the threshold current for both elicited and orthodromically-stimulated action potentials; and prolonged the duration of the orthodromic excitatory post-synaptic potential. These trimethyltin-induced effects were not readily reversed. On the other hand, dimethyltin at high concentrations only reduced the amplitude of the orthodromic action potential. Slice viability was compromised following exposure to trimethyltin, but not following dimethyltin. These data demonstrate that decreased membrane time constant and input resistance is an early and reliable indicator of the onset of trimethyltin-induced changes. The effects do not resemble those produced by excitotoxins, but rather share similarities with responses observed during slice hypoxia.

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

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.