Characteristics of a human N-type calcium channel expressed in HEK293 cells

Bleakman, David; Bowman, D. M. J. S.; Bath, Catherine P.; Brust, Paul F.; Johnson, Edwin C.; Deal, C.; Miller, Richard J.; Ellis, Steven B.; Harpold, Michael M.; Hans, Michael; Grantham, C.J.

doi:10.1016/0028-3908(95)00078-k

Cited by 48 publications

(24 citation statements)

References 24 publications

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“…Ca currents could be elicited in these cells by step depolarizations to different potentials. Cells expressing ␣ 1B produced Ca currents that had properties of typical N currents (Bleakman et al, 1995). Cells expressing ␣ 1E produced currents whose properties we have described previously and which are similar to currents described in the literature as "R" type (Williams et al, 1994;Wheeler et al, 1995).…”

Section: Effects Of Gtp Analogssupporting

confidence: 57%

“…1F). This was presumably attributable to differences in the extent of voltage-dependent inactivation between the two types of Ca channels (Williams et al, 1994;Bleakman et al, 1995). Consistent with this finding, the "R"-type current in cerebellar granule neurons also displayed very pronounced voltage-dependent inactivation (Zhang et al, 1993).…”

Section: Effects Of Gtp Analogsmentioning

confidence: 79%

“…HEK293 cell lines expressing Ca channels with various subunit compositions were kindly provided by SIBIA Inc. (Williams et al, 1992;Bleakman et al, 1995). Briefly, the cell lines were developed by stable co-transfection of HEK293 cells with human ␣ 1 , ␣ 2B ␦, and ␤ Ca channel subunit expression plasmids.…”

Section: Methodsmentioning

confidence: 99%

“…Ca channels in these cells also contained stably expressed ␣ 2 /␦ and ␤ 1B subunits (Williams et al, 1992;Bleakman et al, 1995). It has been shown that the presence of these ancillary subunits is essential for Ca channels to be expressed efficiently and display all of their normal properties (Williams et al, 1992;Bleakman et al, 1995).…”

Section: Effects Of Gtp Analogsmentioning

confidence: 99%

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Selective G-Protein Regulation of Neuronal Calcium Channels

Tóth¹,

Shekter²,

Hui³

et al. 1996

J. Neurosci.

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We examined the properties and regulation of Ca channels resulting from the expression of human ␣ 1B and ␣ 1E subunits stably expressed in HEK293 cells. The ancillary subunits ␤ 1B and ␣ 2 /␦ were also stably expressed in these cell lines. Ca currents in ␣ 1B -expressing cells had the properties of N-type currents. Ca currents in cells expressing ␣ 1E exhibited a novel profile that was similar to the properties of the "R type" Ca current. Introduction of GTP-␥-S into ␣ 1B cells greatly enhanced the extent of prepulse facilitation of the Ca current, whereas it had only a very small effect in ␣ 1E -expressing cells. Activation of somatostatin receptors endogenous to HEK293 cells or opioid receptors, expressed in the cells after transfection, inhibited Ca currents in ␣ 1B -expressing cells. This inhibition was blocked by pertussis toxin and was partially relieved by a depolarizing prepulse. In contrast, no inhibitory effects were noted in cells expressing ␣ 1E channels under the same circumstances. HEK293 cells normally contained G-proteins from all of the four major families. Inhibition of Ca currents by agonists in ␣ 1B -expressing cells was enhanced slightly by the cotransfection of several G-protein ␣ subunits. agonists, however, had no effect in ␣ 1E -containing cells, even after overexpression of different G-protein ␣-subunits. In summary, these results demonstrate that there is a large difference in the susceptibility of ␣ 1B -and ␣ 1E -based Ca channels to regulation by G-proteins. This is so despite the fact that the two types of Ca channels show substantial similarities in their primary sequences.

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Section: Effects Of Gtp Analogssupporting

confidence: 57%

Section: Effects Of Gtp Analogsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Effects Of Gtp Analogsmentioning

confidence: 99%

See 2 more Smart Citations

Selective G-Protein Regulation of Neuronal Calcium Channels

Tóth¹,

Shekter²,

Hui³

et al. 1996

J. Neurosci.

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“…The current in the soma is inhibited in both immature and adult cells by concentrations of -Aga-IVA that are selective for P-type channels (80 -85%, IC 50 Ͻ 1-3 nM) over Q-and N-type channels (Mintz et al, 1992;Sather et al, 1993;Sidach and Mintz, 2000;Tringham et al, 2007) and by -CTx-MVIIC with characteristically slow kinetics (McDonough et al, 1996;Tringham et al, 2007). A paucity of N-type current in the soma of cerebellar Purkinje cells is indicated by the slow kinetics of block by -CTx-MVIIC, by the lack of inhibition in adult Purkinje cells by submicromolar concentrations of -CTx-GVIA (Tringham et al, 2007) that are known to inhibit N-type currents (Boland et al, 1994;Bleakman et al, 1995;Lin et al, 1997), by inhibition of only a minor current component (0 -5%) in dissociated immature cells by -CTx-GVIA (Regan, 1991;Mintz et al, 1992), and by the absence or weak expression of Ca V 2.2 mRNA (Tanaka et al, 1995;Volsen et al, 1995) or Ca V 2.2 protein (Volsen et al, 1995;Chung et al, 2000), although one study did detect Ca V 2.2 protein in Purkinje cells (Westenbroek et al, 1992).…”

mentioning

confidence: 99%

Protease Treatment of Cerebellar Purkinje Cells Renders ω-Agatoxin IVA-Sensitive Ca²⁺ Channels Insensitive to Inhibition by ω-Conotoxin GVIA

Tringham

Dupere²,

Payne³

et al. 2007

J Pharmacol Exp Ther

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The identification of currents carried by N-and P-type Ca 2ϩ channels in the nervous system relies on the use of -conotoxin (CTx) GVIA and -agatoxin (Aga) IVA. The peptide -Aga-IVA inhibits P-type currents at nanomolar concentrations and N-type currents at micromolar concentrations. -CTx-GVIA blocks N-type currents, but there have been no reports that it can also inhibit P-type currents. To assess the effects of -CTx-GVIA on P-type channels, we made patch-clamp recordings from the soma of Purkinje cells in cerebellar slices of mature [postnatal days (P) 40 -50, P40 -50] and immature (P13-20) rats, in which P-type channels carry most of the Ca 2ϩ channel current (Ն85%). These showed that micromolar concentrations of -CTx-GVIA inhibited the current in P40 -50 cells (66%, 3 M; 78%, 10 M) and in P13-20 Purkinje cells (86%, 3 M; 89%, 10 M). The inhibition appeared to be reversible, in contrast to the known irreversible inhibition of N-type current. Exposure of slices from young animals to the enzyme commonly used to dissociate Purkinje cells, protease XXIII, abolished the inhibition by -CTx-GVIA but not by -Aga-IVA (84%, 30 nM). Our finding that micromolar concentrations of -CTx-GVIA inhibit P-type currents suggests that specific block of N-type current requires the use of submicromolar concentrations. The protease-induced removal of block by -CTx-GVIA but not by -Aga-IVA indicates a selective proteolytic action at site(s) on P-type channels with which -CTx-GVIA interacts. It also suggests that Ca 2ϩ channel pharmacology in neurons dissociated using protease may not predict that in neurons not exposed to the enzyme.Voltage-gated Ca 2ϩ channels control numerous cellular functions, including neuronal electrical activity, gene expression, and intracellular signaling. The use of toxins originating from venoms of assorted species has aided the classification of Ca 2ϩ channels as T-, N-, L-, P-, Q-, or R-type and helped elucidate the diverse physiological roles of the different Ca 2ϩ channel classes (McDonough, 2004;McGivern, 2006). It has also helped implicate altered function of distinct types of Ca 2ϩ channels in specific pathophysiological conditions and led to the development of an -conotoxin as an N-type channel blocker that is used in the management of intractable pain (Snutch, 2005;McGivern, 2006).One characteristic by which N-type channels in neurons have been identified and by which recombinant channels containing a Ca V 2.2 subunit have been matched to native N-type channels, is the irreversible inhibition of Ca 2ϩ or Ba 2ϩ currents by -conotoxin (CTx) GVIA (McDonough, 2004;McGivern, 2006). The property used to identify native P-type channels and recombinant channels containing a Ca V 2.1 subunit has been the irreversible inhibition of currents by -agatoxin (Aga) IVA (McDonough, 2004;McGivern, 2006). The selectivity of -Aga-IVA for P-type over N-type channels is not absolute; at concentrations higher than those necessary to block P-type currents, -Aga-IVA also blocks N-type currents (Sidach and Mintz, 20...

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