Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.     

A novel Ca(2+) influx pathway in mammalian primary sensory neurons is activated by caffeine.

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.     

Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons

Mudpuppy parasympathetic neurons exhibit spontaneous miniature hyperpolarizations (SMHs) that are generated by potassium currents, which are spontaneous miniature outward currents (SMOCs), flowing through clusters of large conductance voltage- and calcium (Ca(2+))-activated potassium (BK) channels. The underlying SMOCs are initiated by a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Perforated-patch whole cell voltage recordings were used to determine whether activation of SMHs contributed to action potential (AP) repolarization or affected the latency to AP generation. Blockade of BK channels by iberiotoxin (IBX, 100 nM) slowed AP repolarization and increased AP duration. Treatment with omega-conotoxin GVIA (3 microM) or nifedipine (10 microM) to inhibit Ca(2+) influx through N- or L-type voltage-dependent calcium channels (VDCCs), respectively, also decreased the rate of AP repolarization and increased AP duration. Elimination of CICR by treatment with either thapsigargin (1 microM) or ryanodine (10 microM) produced no significant change in AP repolarization or duration. Blockade of BK channels with IBX and inhibition of N-type VDCCs with omega-conotoxin GVIA, but not inhibition of L-type VDCCs with nifedipine, decreased the latency of AP generation. A decrease in latency to AP generation occurred with elimination of SMHs by inhibition of CICR following treatment with thapsigargin. Ryanodine treatment decreased AP latency in three of six cells. Apamin (100 nM) had no affect on AP repolarization, duration, or latency to AP generation, but did decrease the hyperpolarizing afterpotential (HAP). Inhibition of L-type VDCCs by nifedipine also decreased HAP amplitude. Inhibition of CICR by either thapsigargin or ryanodine treatment increased the number of APs generated with long depolarizing current pulses, whereas exposure to IBX or omega-conotoxin GVIA depressed excitability. We conclude that CICR, the process responsible for SMH generation, represents a unique mechanism to modulate the response to subthreshold depolarizing currents that drive the membrane potential toward the threshold for AP initiation but does not contribute to AP repolarization. Subthreshold depolarizations would not activate sufficient numbers of VDCCs to allow Ca(2+) influx to elevate [Ca(2+)](i) to the extent needed to directly activate nearby BK channels. However, the elevation in [Ca(2+)](i) is sufficient to trigger CICR from ryanodine-sensitive Ca(2+) stores. Thus CICR acts as an amplification mechanism to trigger a local elevation of [Ca(2+)](i) near a cluster of BK channels to activate these channels at negative levels of membrane potential.     

Activation of BK channels in rat chromaffin cells requires summation of Ca(2+) influx from multiple Ca(2+) channels

Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at a distance of 50 to 160 nm from Ca(2+) channels. A model that assumes random distribution of Ca(2+) and BK channels fails to account for the observed [Ca(2+)](i) detected by BK channels, suggesting that a specific mechanism may exist to mediate the functional coupling between these channels. Importantly, the effects of EGTA and BAPTA cannot be explained by assuming a one-to-one coupling between Ca(2+) and BK channels. Rather, Ca(2+) influx through a number of Ca(2+) channels appears to act in concert to regulate the behavior of any individual BK channel. Thus differences in BK channel open probabilities may be explained by differences in the extent of Ca(2+) domain overlap at the sites of individual BK channels.     

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Diversity of channels involved in Ca(2+) activation of K(+) channels during the prolonged AHP in guinea-pig sympathetic neurons

The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)-dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2+) entry through L-type Ca(2+) channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N-type Ca(2+) channels (with 100 nM omega-conotoxin GVIA) and P-type Ca(2+) channels (with 40 nM omega-agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca(2+) influx through multiple types of voltage-gated Ca(2+) channel can activate the intracellular mechanisms that generate gKCa2. The slow time course of gKCa2 may be explained if activation of multiple K(+) channels results from Ca(2+) influx triggering a kinetically invariant release of Ca(2+) from intracellular stores located close to the membrane.     

Mitral cell presynaptic Ca(2+) influx and synaptic transmission in frog amygdala

Dextran-conjugated Ca(2+) indicators were injected into the accessory olfactory bulb of frogs in vivo to selectively fill presynaptic terminals of mitral cells at their termination in the ipsilateral amygdala. After one to three days of uptake and transport, the forebrain hemisphere anterior to the tectum was removed and maintained in vitro for simultaneous electrophysiological and optical measurements. Ca(2+) influx into these terminals was compared to synaptic transmission between mitral cells and amygdala neurons under conditions of reduced Ca(2+) influx resulting from reduced extracellular [Ca(2+)], blockade of N- and P/Q-type channels, and application of the cholinergic agonist carbachol. Reducing extracellular [Ca(2+)] had a non-linear effect on release; release was proportional to Ca(2+) influx raised to the power of approximately 3.6, as observed at numerous other synapses. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1 microM), blocked 77% of Ca(2+) influx and 88% of the postsynaptic field potential. The P/Q-type Ca(2+) channel blocker, omega-agatoxin-IVA (200 nM), blocked 19% of Ca(2+) influx and 25% of the postsynaptic field, while the two toxins combined to block 92% of Ca(2+) influx and 97% of the postsynaptic field. The relationship between toxin blockade of Ca(2+) influx and synaptic transmission was therefore only slightly non-linear; release was proportional to Ca(2+) influx raised to the power approximately 1.4. Carbachol (100 microM) acting via muscarinic receptors had no effect on the afferent volley, but rapidly and reversibly reduced Ca(2+) influx through both N- and P/Q-type channels by 51% and postsynaptic responses by 78%, i.e. release was proportional to Ca(2+) raised to the power approximately 2.5.The weak dependence of release on changes in Ca(2+) when channel toxins block channels suggests little overlap between Ca(2+) microdomains from channels supporting release or substantial segregation of channel subtypes between terminals. The proportionately greater reduction of transmission by muscarinic receptors compared to Ca(2+) channel toxins suggests that they directly affect the release machinery in addition to reducing Ca(2+) influx.     

Mitochondrial modulation of Ca2+ -induced Ca2+ -release in rat sensory neurons

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.     

Ca2+ influx, but not Ca2+ release from internal stores, is required for the PACAP-induced increase in excitability in guinea pig intracardiac neurons

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.     

A store-operated Ca(2+) influx pathway in the bag cell neurons of Aplysia

Although store-operated Ca(2+) influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca(2+) entry pathway can be activated by Ca(2+) store depletion. Using fura-based imaging of intracellular Ca(2+) in cultured bag cell neurons, we now characterize this pathway as store-operated Ca(2+) influx. In the absence of extracellular Ca(2+), the endoplasmic reticulum Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca(2+). With the subsequent addition of extracellular Ca(2+), a prominent Ca(2+) influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca(2+) but did not initiate store-operated Ca(2+) influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H(+)-ATPase inhibitor, liberated intracellular Ca(2+) from acidic stores and attenuated subsequent Ca(2+) influx, presumably by replenishing CPA-depleted stores. Store-operated Ca(2+) influx was partially blocked by low concentrations of La(3+) or BTP2, and strongly inhibited by either 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni(2+). Regarding IP(3) receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca(2+) influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca(2+) release and neuropeptide secretion. Store-operated Ca(2+) influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior.     

IP(3)-Independent release of Ca(2+) from intracellular stores: A novel mechanism for transduction of bitter stimuli

A variety of substances with different chemical structures elicits a bitter taste. Several different transduction mechanisms underlie detection of bitter tastants; however, these have been described in detail for only a few compounds. In addition, most studies have focused on mammalian taste cells, of which only a small subset is responsive to any particular bitter compound. In contrast, approximately 80% of the taste cells in the mudpuppy, Necturus maculosus, are bitter-responsive. In this study, we used Ca(2+) imaging and giga-seal whole cell recording to compare the transduction of dextromethorphan (DEX), a bitter antitussive, with transduction of the well-studied bitter compound denatonium. Bath perfusion of DEX (2.5 mM) increased the intracellular Ca(2+) level in most taste cells. The DEX-induced Ca(2+) increase was inhibited by thapsigargin, an inhibitor of Ca(2+) transport into intracellular stores, but not by U73122, an inhibitor of phospholipase C, or by ryanodine, an inhibitor of ryanodine-sensitive Ca(2+) stores. Increasing intracellular cAMP levels with a cell-permeant cAMP analogue and a phosphodiesterase inhibitor enhanced the DEX-induced Ca(2+) increase, which was inhibited partially by H89, a protein kinase A inhibitor. Electrophysiological measurements showed that DEX depolarized the membrane potential and inhibited voltage-gated Na(+) and K(+) currents in the presence of GDP-beta-S, a blocker of G-protein activation. DEX also inhibited voltage-gated Ca(2+) channels. We suggest that DEX, like quinine, depolarizes taste cells by block of voltage-gated K channels, which are localized to the apical membrane in mudpuppy. In addition, DEX causes release of Ca(2+) from intracellular stores by a phospholipase C-independent mechanism. We speculate that the membrane-permeant DEX may enter taste cells and interact directly with Ca(2+) stores. Comparing transduction of DEX with that of denatonium, both compounds release Ca(2+) from intracellular stores. However, denatonium requires activation of phospholipase C, and the mechanism results in a hyperpolarization rather than a depolarization of the membrane potential. These data support the hypothesis that single taste receptor cells can use multiple mechanisms for transducing the same bitter compound.     

Dopamine modulates exocytosis independent of Ca(2+) entry in melanotropic cells

Dopamine is a known inhibitor of pituitary melanotropic cells. It reduces Ca(2+) influx by hyperpolarizing the cell membrane and by modulating high- and low-voltage-activated (HVA and LVA) Ca(2+) channels. As a result, dopamine reduces the hormonal output of the cell. However, it is unknown how dopamine affects each of the four different HVA Ca(2+) channel types individually. Moreover, it is unknown whether dopamine interacts with exocytosis independent of Ca(2+) channels. Here we show that dopamine differentially modulates the HVA Ca(2+) channels and that it affects the stimulus-secretion coupling through a direct effect on the exocytotic machinery. Sustained L- and P-type Ba(2+) currents are reduced in amplitude and inactivating N- and Q-type currents acquire different activation and inactivation kinetics in the presence of dopamine. The Q-type current shows slow activation, which is a hallmark for direct G-protein modulation. We used membrane capacitance measurements to monitor exocytosis. Surprisingly, we find that the amount of exocytosis per step depolarization is not diminished by dopamine despite the reduction in Ca(2+) current. To test whether dopamine affects the release machinery downstream of Ca(2+) entry, we stimulated exocytosis by dialyzing cells with buffered high-Ca(2+) solutions. Dopamine increased the amount and the rate of exocytosis. In the first 90 s, the rate of secretion was increased two- to threefold, but it was normalized again at 180 s, suggesting that predominantly vesicles that fuse early in the exocytotic phase are modulated by dopamine. Thus while Ca(2+) channels are inhibited by dopamine, the exocytotic machinery downstream of Ca(2+) influx is sensitized. As a result, release is more effectively stimulated by Ca(2+) influx during dopamine inhibition.     

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

We studied inactivation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca(2+)](i) due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca(2+) entry and little depletion of Ca(2+) stores. The Ca(2+) entry elicited by the subsequent pulse within 50 ms, during which the [Ca(2+)](i) level remained unchanged, did not generate a distinct [Ca(2+)](i) rise. This mode of [Ca(2+)](i) rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 microm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct >or= 50 ms after depolarization and depended on [Ca(2+)](i). The inactivation was prevented by BAPTA (>or= 100 microm) but not by EGTA (相似文献   

T-type Ca2+ channels contribute to IBMX/forskolin- and K(+)-induced Ca(2+) transients in porcine olfactory receptor neurons

T-type Ca(2+) channels are low-voltage-activated Ca(2+) channels that control Ca(2+) entry in excitable cells during small depolarization above resting potentials. Using Ca(2+) imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca(2+) channels in IBMX/forskolin- and sparingly elevated extracellular K(+)-induced Ca(2+) transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10microM) or Ni(2+) (100microM), the selective T-type Ca(2+) channel inhibitors, IBMX/forskolin-induced Ca(2+) transients in the soma were either strongly (>60%) inhibited or abolished completely. However, the Ca(2+) transients in the knob were only partially (<60%) inhibited. Ca(2+) transients induced by 30mM K(+) were also partially ( approximately 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5mM increase in the extracellular K(+) concentration (7.5mM K(+)), and such responses were completely inhibited by mibefradil or Ni(2+). These results reveal functional expression of T-type Ca(2+) channels in porcine ORNs, and suggest a role for these channels in the spread Ca(2+) transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.     

Increase in intracellular Ca(2+) and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca(2+) concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 microm) loaded with fura-2, a bath application of ATP (100 microM) evoked an increase in intracellular Ca(2+) concentration, while the removal of extracellular Ca(2+) partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca(2+) concentration is due to both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). Bath application of uridine 5'-triphosphate (UTP; 100 microM) also caused an increase in intracellular Ca(2+) concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 microM) virtually abolished the response, indicating that the intracellular Ca(2+) elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca(2+) increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca(2+) is primarily due to the release of Ca(2+) from endoplasmic reticulum Ca(2+) stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 microM) significantly stimulated the release of CGRP and that suramin (100 microM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca(2+) is accompanied by CGRP release from dorsal root ganglion neurons.These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca(2+) concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.     

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells.

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 microM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.     

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.     

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.