1. The effects of metabotropic glutamate receptor (mGluR) stimulation on whole-cell Ca2+ currents were studied in pyramidal neurons isolated from the dorsal frontoparietal neocortex of rat. The selective mGluR agonist cis-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid [trans-ACPD (100 microM)] suppressed the peak high-threshold Ca2+ current by 21 +/- 1.7% (mean +/- SE) in 40 of 43 cells from 10- to 21-day-old rats. Consistent with previous findings for mGluR, glutamate, quisqualate, and ibotenate [but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)] reduced the Ca2+ currents, and the responses were not blocked by the ionotropic glutamate receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovaleric acid (APV). EC50S for Ca2+ current suppression were 29 nM for quisqualate, 2.3 microM for glutamate, and 13 microM for trans-ACPD. 2. The low-threshold Ca2+ current was not modulated by trans-ACPD. The component of the high-threshold CA2+ current suppressed by mGluR was determined by pharmacology; the responses were not affected by omega-conotoxin GVIA but were occluded by the dihydropyridine Ca2+ antagonist nifedipine. Ca2+ tail currents prolonged by the dihydropyridine Ca2+ agonist (+)-SDZ 202-79] were suppressed by mGluR stimulation in parallel with the peak current. These findings strongly suggest that L-type Ca2+ channels are modulated by mGluR. 3. In neurons dialyzed with 100 microM guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S), Ca2+ current suppression was elicited by the first application of trans-ACPD (in 5 of 6 cells), but not by subsequent applications. Responses in neurons dialyzed with 2 mM guanosine 5'-(beta-thio)diphosphate (GDP-beta-S) were significantly smaller than controls. The results are consistent with mGluR acting via linkage to a G protein. 4. The responses to mGluR agonists were smaller when the external Ca2+ was replaced by Ba2+, indicating that some part of the mechanism underlying the current suppression is Ca2+ dependent. Because mGluR stimulates phosphoinositide turnover and release of Ca2+ from intracellular stores in other types of neurons, the possibility of released Ca2+ mediating inactivation of Ca2+ channels was considered. However, the Ca2+ current suppression was not attenuated by strong intracellular Ca2+ buffering [20 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)], by dialysis with 100 microM inositol-1,4,5-triphosphate (IP3), or by external application of 1 microM thapsigargin. 5. We conclude that in neocortical neurons, one action of mGluR is to suppress the component of high-threshold Ca2+ current conducted by L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

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1. Ca2+ currents were investigated in neurons acutely isolated from adult human temporal neocortex. The aim was to compare the basic characteristics of the currents with those previously described in animals and to examine the effects of dihydropyridine Ca2+ antagonists and antiepileptic drugs. The tissue, obtained from patients undergoing temporal lobe surgery for medically intractable epilepsy, was sliced, incubated in papain, and triturated. 2. Most of the isolated neurons (34 of 36) were judged to be pyramidal cells by their morphology. Whole-cell voltage-clamp recordings revealed two components of Ca2+ current: 1) a low-threshold (T-type) current that was transient, small in amplitude, and required hyperpolarization more negative than -70 mV for removal of inactivation and 2) a high-threshold current that was slowly inactivating and was available for activation from more positive potentials. The characteristics of the Ca2+ currents were very similar to those in the neocortical neurons of young rats, although the low-threshold current was less prominent in the human cells. 3. Subcomponents of the high-threshold current were identified by pharmacology. About 20% of the peak current was blocked by omega-conotoxin GVIA (presumed N current) and 40-50% of the peak current was blocked by micromolar concentrations of the dihydropyridine Ca2+ antagonists nifedipine and nimodipine (presumed L current). In two neurons tested with a range of nimodipine concentrations, the threshold for suppression of the high-threshold current was approximately 10 nM. 4. The antiepileptic agents ethosuximide, carbamazepine, and valproate did not affect the Ca2+ currents at therapeutically relevant concentrations. Phenytoin marginally reduced the low- and high-threshold Ca2+ currents at 8 microM (a concentration corresponding to the upper therapeutic range). The results do not support the hypothesis that inhibition of Ca2+ currents in neocortical pyramidal neurons is a major action of these drugs.

ERp29 is a recently discovered resident of the endoplasmic reticulum (ER) that is abundant in brain and most other mammalian tissues. Investigations of nonneural secretory tissues have implicated ERp29 in a major role producing export proteins, but a molecular activity remains wanting for this functional orphan. Intriguingly, ERp29 appears to be heavily utilized in the cerebellum, a brain region not conventionally regarded as neurosecretory. To elucidate this functional quandary, we used immunochemical approaches to characterize the regional, cellular, and subcellular distributions of ERp29 in rat brain. Immunohistochemistry revealed ubiquitous expression in neuronal and nonneuronal cells, with a distinctive variation in somatic ERp29 levels. Highly expressing cells were found in diverse locations, implying that ERp29 is not biased towards the cerebellum functionally. Using immunolocalization data mined from the literature, a proteomic profile was developed to assess the functional significance of ERp29's characteristic expression pattern. Surprisingly, ERp29 correlated poorly with classical markers of neurosecretion, but strongly with a variety of major membrane proteins. Together with immunogold localization of ERp29 to somatic ER, these observations led to a novel hypothesis that ERp29 is involved primarily in production of endomembrane proteins rather than proteins destined for export. This study establishes ERp29 as a general ER marker for brain cells and provides a stimulating clue about ERp29's enigmatic function. ERp29 appears to have broad significance for neural pathophysiology, given its ubiquitous distribution and prominence in brain over classical ER residents like BiP and protein disulfide isomerase.

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin 28kDa . Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin 28kDa -null mutants. An unchanged rate of calcium transport was verified by measurements of 45 Ca incorporation into developing teeth in vivo. In enamelforming cells, the absence of calbindin 28kDa was not compensated by other cytosolic calcium-binding proteins as detectable by 45 Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.The active transport of calcium across cells holds widespread importance in medicine and biology, yet the underlying mechanisms remain unclear. Operating in many places (e.g. gut, kidney, placenta, teeth, bones, oviduct, lung, inner ear), active transport is used to control the amount of calcium in body fluids and so impacts on nutrition, biomineralization, fertility, respiration, and hearing (1, 2). Superior control is achieved by passaging calcium actively through cells rather than passively between them, but this comes at the risk of cytotoxicity should the ability to regulate intracellular calcium be overburdened.Mechanistically, active transport is considered in three steps: the entry of calcium to the cell, transit across it, and extrusion at the other side. The transit step has received the most attention over several decades, being considered rate-limiting and having key molecular players defined. However, recent molecular characterization of calcium entry channels has transformed the field by providing a new mechanistic focus for vitamin D-restricted transport (3, 4). With these advances reigniting interest in therapeutic applications, it is important to revisit what happens following calcium entry.The 30-year-old paradigm that calcium is ferried through cytosol by mobile calcium-binding proteins (calbindins) remains widely accepted (3-9). Calbindins are thought to facilitate the naturally poor diffusion of calcium in cytosol and simultaneously buffer calcium at safe concentrations. Comprehensively supporting this view, tight correlations between calbindin expression and vitamin D-dependent transport were found in intestine...