Related items
Showing items related by title, author, creator and subject.
-
Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminalsCa2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.
-
Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ releaseSelective 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.
-
Channel Specific Calcium Dynamics in PC12 Cells: A DissertationCalcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.
