A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry.

The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+-dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+-channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bbeta3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 microM FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+-dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mM BAPTA or 1 microM FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels.

Overlapping selectivity of neurotoxin and dihydropyridine calcium channel blockers in cerebellar granule neurones.

Calcium (Ca(2+)) currents have been studied extensively in cerebellar granule neurones, but much of the whole-cell pharmacology is inconsistent. Ca(2+) channel currents were recorded from granule neurones to investigate whether the commonly used Ca(2+) channel blockers show overlapping selectivity. Using combinations of toxin channel blockers, 45% of the total current was shown to be carried by Ca(2+) channels susceptible to block by the combined, or cumulative application of, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC, thus representing P/Q- and N-type channel currents. However, sequential application of these toxins showed that substantial overlap occurred in the proportions of current sensitive to individual toxins. Application of the 1, 4-dihydropyridine nicardipine at 1 microM, a concentration reported to be selective for L-type channels, blocked 16% of the total current, without reducing the current sensitive to the toxins used. However, greater concentrations of nicardipine (>10 microM) blocked a proportion of the total current that could not be accounted for by L-type channels alone. These results demonstrate that a pharmacological approach based on the L, N, P/Q, and R classification does not adequately describe the Ca(2+) channel subtypes found in cerebellar granule neurones due to substantial cross-selectivity to the drugs and toxins used.

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.

In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.