Faculty Bibliography

This study describes a Ca2+ store in fura-2-loaded bullfrog sympathetic neurons that modulates [Ca2+]i responses elicited by either depolarization or Ca2+ release from a caffeine- and ryanodine-sensitive store. This store is insensitive to caffeine and ryanodine, but is sensitive to the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The FCCP-sensitive store slows both the rise in [Ca2+]i during stimulation (apparently by accumulating Ca2+ from the cytosol) and the recovery following stimulation (by releasing the accumulated Ca2+ into the cytosol). For a fixed level of depolarization, recovery is slowed to an extent that depends on stimulus duration. [Ca2+]i imaging shows that these effects are prominent in the soma but not in growth cones. Ca2+ uptake by the FCCP-sensitive store appears to be strongly [Ca2+]i dependent, since it becomes influential only when [Ca2+]i approaches approximately 500 nM. Therefore, this store may specifically influence [Ca2+]i during moderate and strong stimulation. The effect of the store on responses to depolarization can be accounted for by a simple three-compartment scheme consisting of the extracellular medium, the cytosol, and a single internal store with a [Ca2+]i-dependent uptake mechanism resembling the mitochondrial Ca2+ uniporter. The store's effect on responses to caffeine-induced Ca2+ release can be accounted for by including a second internal compartment to represent the caffeine-sensitive store. While the identity of the FCCP-sensitive store is unknown, its sensitivity to FCCP is consistent with a mitochondrial pool. It is suggested that by modulating the temporal properties of [Ca2+]i following stimulation, the FCCP-sensitive store may influence the degree of activation of intracellular [Ca2+]i-dependent processes

Neurotoxins that selectively block Na+, K+ or Ca2+ channels have provided valuable information about the functional diversity of the voltage-gated channel superfamily. For Ca2+ channels, a variety of toxins have been found to block individual channel types. The best-known example is omega-conotoxin-GVIA, a member of a large family of peptide toxins derived from venomous cone snails, which potently and selectively blocks N-type Ca2+ channels, allowing their purification, cellular localization, and the elucidation of their roles in Ca2+ entry, neurotransmitter release and neuronal migration. In contrast to Na+ and K+ channels, little is known about the molecular features that underlie Ca(2+)-channel susceptibility to toxin block; it is also unknown whether block occurs by direct physical occlusion or an action on channel gating. Here we describe structural determinants of N-type Ca2+ channel's interaction with omega-conotoxin-GVIA. When chimaeras combining individual motifs from the N-type channel and from a channel insensitive to omega-conotoxin-GVIA were expressed in Xenopus oocytes, each of the four motifs appeared to contribute to interaction with the toxin. The most dramatic effects on toxin interactions were seen at a single cluster of residues in the large putative extracellular loop between IIIS5 and IIIH5, consistent with a direct pore-blocking mechanism. These results provide a starting point for delineating the architecture of the outer vestibule of the Ca2+ channel

It has been proposed that nitric oxide (NO) serves as a key retrograde messenger during long-term potentiation at hippocampal synapses, linking induction of long-term potentiation in postsynaptic CA1 pyramidal cells to expression of long-term potentiation in presynaptic nerve terminals. However, nitric oxide synthase (NOS), the proposed NO-generating enzyme, has not yet been detected in the appropriate postsynaptic cells. We here demonstrate specific NOS immunoreactivity in the CA1 region of hippocampal sections by using an antibody specific for NOS type I and relatively gentle methods of fixation. NOS immunoreactivity was found in dendrites and cell bodies of CA1 pyramidal neurons. Cultured hippocampal pyramidal cells also displayed specific immunostaining. Control experiments showed no staining with preimmune serum or immune serum that was blocked with purified NOS. These results demonstrate that CA1 pyramidal cells contain NOS, as required were NO involved in retrograde signaling during hippocampal synaptic plasticity

Several types of calcium channels found in the central nervous system are possible participants in triggering neurotransmitter release. Synaptic transmission between hippocampal CA3 and CA1 neurons was mediated by N-type calcium channels, together with calcium channels whose pharmacology differs from that of L- and P-type channels but resembles that of the Q-type channel encoded by the alpha 1A subunit gene. Blockade of either population of channels strongly increased enhancement of synaptic transmission with repetitive stimuli. Even after complete blockade of N-type channels, transmission was strongly modulated by stimulation of neurotransmitter receptors or protein kinase C. These findings suggest a role for alpha 1A subunits in synaptic transmission and support the idea that neurotransmitter release may depend on multiple types of calcium channels under physiological conditions

There has been rapid progress in understanding the structural basis of ion selectivity and permeation in both ligand- and voltage-gated channels. Recognition of similarities in overall architecture within a channel class has led to an increasing focus on the specific molecular determinants that endow a channel with its own distinctive character. It has been possible in some cases to identify individual amino acids essential for ion selectivity

Voltage-dependent Ca2+ channels respond to membrane depolarization by conformational changes that control channel opening and eventual closing by inactivation. The kinetics of inactivation differ considerably between types of Ca2+ channels and are important in determining the amount of Ca2+ entry during electrical activity and its resulting impact on diverse cellular events. The most intensively characterized forms of inactivation in potassium and sodium channels involve pore block by a tethered plug. In contrast, little is known about the molecular basis of Ca(2+)-channel inactivation. We studied the molecular mechanism of inactivation of voltage-gated calcium channels by making chimaeras from channels with different inactivation rates. We report here that the amino acids responsible for the kinetic differences are localized to membrane-spanning segment S6 of the first repeat of the alpha 1 subunit (IS6), and to putative extracellular and cytoplasmic domains flanking IS6. Involvement of this region in Ca(2+)-channel inactivation was unexpected and raises interesting comparisons with Na+ channels, where the III-IV loop is a critical structural determinant. Ca(2+)-channel inactivation has some features that resemble C-type inactivation of potassium channels

In many neurons, N-type Ca2+ channels are a major Ca2+ entry pathway and strongly influence neurotransmitter release. We carried out cell-attached patch recordings (110 mM Ba2+ as charge carrier) to characterize the rapid opening and closing kinetics of N-type Ca2+ channel gating in frog sympathetic neurons. Single channels display at least three distinct patterns of gating, characterized as low-, medium-, and high-rho o modes on the basis of channel open probability (rho o) during depolarizing pulses to -10 mV. Spontaneous transitions from one mode to another are infrequent, with an exponential distribution of dwell times and mean sojourns of approximately 10 sec in each mode. Thus, a channel typically undergoes hundreds or thousands of open-closed transitions in one mode before switching to a different mode. Transitions between modes during a depolarization were occasionally detected, but were rare, as expected for infrequent modal switching. Within each mode, the activation kinetics were well described by a simple scheme (C2-C1-O), as previously reported for other types of Ca2+ channels. Rate constants are strikingly different from one mode to another, giving each mode its own characteristic kinetic signature. The gating behavior at -10 mV ranges from brief openings (approximately 0.3 msec) and long closures (10-20 msec) for low-rho o gating to long openings (3 msec) and brief closures (approximately 1 msec) for high-rho o gating. Intermediate values for mean open durations (approximately 1.5 msec) and mean closed durations (approximately 3 msec) were found for medium-rho o gating. In addition to being kinetically distinct, channel openings in the low-rho o mode often exhibit a unitary current approximately 0.2 pA larger than in the medium- or high-rho o mode. Each mode is characterized by its own voltage dependence: activation occurs at relatively negative potentials and is most steeply voltage dependent in the high-rho o mode, while activation requires very strong depolarizations and is weakly voltage dependent in the low-rho o mode. The proportion of time spent in the individual modes varies greatly from one patch to another, suggesting that modal gating may be subject to cellular control