The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog's sacculus.

Neurotransmitter is released when Ca2+ triggers the fusion of synaptic vesicles with the plasmalemma. To study factors that regulate Ca2+ concentration at the presynaptic active zones of hair cells, we used laser-scanning confocal microscopy with the fluorescent Ca2+ indicator fluo 3. The experimental results were compared with the predictions of a model of presynaptic Ca2+ concentration in which Ca2+ enters a cell through a point source, diffuses from the entry site, and binds to fixed or mobile Ca2+ buffers. The observed time course and magnitude of fluorescence changes under a variety of conditions were well fit when the model included mobile molecules as the only Ca2+ buffer. The results confirm the localized entry of Ca2+ underlying neurotransmitter release and suggest that Ca2+ is cleared from an active zone almost exclusively by mobile buffer.

Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+.

Classical conditioning of Aplysia's siphon-withdrawal reflex is thought to be due to a presynaptic mechanism-activity-dependent presynaptic facilitation of sensorimotor connections. Recent experiments with sensorimotor synapses in dissociated cell culture, however, provide an alternative cellular mechanism for classical conditioning-Hebbian long-term potentiation (LTP) of sensorimotor connections. Induction of Hebbian LTP of these connections is mediated by activation of N-methyl-D-aspartate-related receptors and requires the postsynaptic elevation of intracellular Ca2+. To determine whether the enhancement of sensorimotor synapses during classical conditioning in Aplysia-like LTP of sensorimotor synapses in culture-also depends upon the elevation of postsynaptic Ca2+, we carried out experiments involving the cellular analog of classical conditioning of siphon withdrawal. We examined changes in the strength of monosynaptic siphon sensorimotor connections in the abdominal ganglion of Aplysia following paired presentations of sensory neuron activation and tail nerve shock. This training regimen resulted in significant enhancement of the monosynaptic sensorimotor excitatory postsynaptic potential, as compared with the sensorimotor excitatory postsynaptic potential in preparations that received only test stimulation. Infusing the motor neuron with 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid, a specific chelator of intracellular Ca2+, prior to paired stimulation training blocked this synaptic enhancement. Our results implicate a postsynaptic, possibly Hebbian, mechanism in classical conditioning in Aplysia.

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

CAX1, an H+/Ca2+ antiporter from Arabidopsis.

Reestablishment of the resting state after stimulus-coupled elevations of cytosolic-free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. Here we describe the isolation of two genes, CAX1 and CAX2, from Arabidopsis thaliana that suppress a mutant of Saccharomyces cerevisiae that has a defect in vacuolar Ca2+ accumulation. Both genes encode polypeptides showing sequence similarities to microbial H+/Ca2+ antiporters. Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAX1 or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAX1 gene product indicate that it is the high capacity transporter responsible for maintaining low cytosolic-free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation.

Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.

The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B.

In the structure of bovine mitochondrial F1-ATPase that was previously determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. Adenylyl-imidodiphosphate is bound to one beta-subunit (betaTP), ADP is bound to the second (betaDP), and no nucleotide is bound to the third (betaE). Here we show that the uncompetitive inhibitor aurovertin B binds to bovine F1 at two equivalent sites in betaTP and betaE, in a cleft between the nucleotide binding and C-terminal domains. In betaDP, the aurovertin B pocket is incomplete and is inaccessible to the inhibitor. The aurovertin B bound to betaTP interacts with alpha-Glu399 in the adjacent alphaTP subunit, whereas the aurovertin B bound to betaE is too distant from alphaE to make an equivalent interaction. Both sites encompass betaArg-412, which was shown by mutational studies to be involved in binding aurovertin. Except for minor changes around the aurovertin pockets, the structure of bovine F1-ATPase is the same as determined previously. Aurovertin B appears to act by preventing closure of the catalytic interfaces, which is essential for a catalytic mechanism involving cyclic interconversion of catalytic sites.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants.

A group of resident ER proteins have been identified that are proposed to function as molecular chaperones. The best characterized of these is BiP/GRP78, an hsp70 homologue that binds peptides containing hydrophobic residues in vitro and unfolded or unassembled proteins in vivo. However, evidence that mammalian BiP plays a direct role in protein folding remains circumstantial. In this study, we examine how BiP interacts with a particular substrate, immunoglobulin light chain (lambda LC), during its folding. Wild-type hamster BiP and several well-characterized BiP ATPase mutants were used in transient expression experiments. We demonstrate that wild-type lambda LCs showed prolonged association with mutant BiP which inhibited their secretion. Both wild-type and mutant BiP bound only to unfolded and partially folded LCs. The wild-type BiP was released from the incompletely folded LCs, allowing them to fold and be secreted, whereas the mutant BiP was not released. As a result, the LCs that were bound to BiP mutants were unable to undergo complete disulfide bond formation and were retained in the ER. Our experiments suggest that LCs undergo both BiP-dependent and BiP-independent folding steps, demonstrating that both ATP binding and hydrolysis activities of BiP are essential for the completion of LC folding in vivo and reveal that BiP must release before disulfide bond formation can occur in that domain.

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).