CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

Mudado, M.A.; Rodrigues, Andreia Laura Prates; Prado, Vânia F.; Beirão, Paulo Sérgio Lacerda; Cruz, Jáder Santos

doi:10.1590/s0100-879x2004000600020

Cited by 11 publications

(7 citation statements)

References 19 publications

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“…Likewise, the use of primers for the Ca V 3.3 sequence did not result in the production of a specific product from GH 3 cells. These results corroborate previous reports showing that one of the three isoforms (Ca V 3.1) is expressed in the GH 3 cells (Glassmeier et al 2001;Mudado et al 2004). In contrast to our findings, Mudado et al (2004) reported the expression of Ca V 3.3 channels in the GH 3 cells, however their PCR experiments revealed the presence of multiple hybridization bands with the Ca V 3.3 oligonucleotides, and no PCR products were apparently sequenced.…”

Section: Resultssupporting

confidence: 91%

“…These results corroborate previous reports showing that one of the three isoforms (Ca V 3.1) is expressed in the GH 3 cells (Glassmeier et al 2001;Mudado et al 2004). In contrast to our findings, Mudado et al (2004) reported the expression of Ca V 3.3 channels in the GH 3 cells, however their PCR experiments revealed the presence of multiple hybridization bands with the Ca V 3.3 oligonucleotides, and no PCR products were apparently sequenced. In order to investigate the expression of the LVA channels at the level of protein, membranes derived from GH 3 cells were screened for the presence of Ca V 3 channels using specific antibodies.…”

Section: Resultssupporting

confidence: 91%

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Regulation of CaV3.1 Channels by Glucocorticoids

et al. 2009

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The activity of low voltage-activated Ca(2+) (Ca(V)3) channels is tightly coupled to neurotransmitter and hormone secretion. Previous studies have shown that Ca(V)3 channels are regulated by glucocorticoids (GCs), though the mechanism underlying channel regulation remains unclear. Here, using the pituitary GH(3) cell line as a model, we investigated whether Ca(V)3 channel expression is under the control of GCs, and if their actions are mediated by transcriptional and/or post-transcriptional mechanisms. RT-PCR and western blot analyses showed that Ca(V)3.1 but not Ca(V)3.2 and Ca(V)3.3 channels is expressed in the GH(3) cells, and patch clamp recordings confirmed that Ca(2+) currents through low voltage-activated channels were decreased after chronic treatment with GCs. Consistent with this, total plasma membrane expression of Ca(V)3.1 protein as analyzed by cell-surface biotinylation assays and semi-quantitative western blotting was also down-regulated, while quantitative real-time RT-PCR analysis revealed a significant decrease of Ca(V)3.1 mRNA expression in the treated cells. In contrast, patch-clamp recordings on HEK-293 cells stably expressing recombinant Ca(V)3.1 channels showed that Ca(2+) currents were not affected by GC treatment. These results suggest that decreased transcription is a likely mechanism to explain the inhibitory actions of GCs on the functional expression of native Ca(V)3.1 channels.

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 91%

Regulation of CaV3.1 Channels by Glucocorticoids

et al. 2009

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“…The Ca v 3.1 and Ca v 3.3 mRNAs were detected in GH 3 cells exhibiting prominent T-type Ca 2ϩ current (105). Several pore-forming subunits of Ca v channels are present in GH 3 /B 6 pituitary cells and account for the formation of T-type (Ca v 3.1), L-type (Ca v 1.1, 1.2, and 1.3), and P/Q (Ca v 2.1) type currents.…”

Section: Voltage-gated Ca 2؉ Channelsmentioning

confidence: 94%

Ion Channels and Signaling in the Pituitary Gland

2010

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Endocrine pituitary cells are neuronlike; they express numerous voltage-gated sodium, calcium, potassium, and chloride channels and fire action potentials spontaneously, accompanied by a rise in intracellular calcium. In some cells, spontaneous electrical activity is sufficient to drive the intracellular calcium concentration above the threshold for stimulus-secretion and stimulus-transcription coupling. In others, the function of these action potentials is to maintain the cells in a responsive state with cytosolic calcium near, but below, the threshold level. Some pituitary cells also express gap junction channels, which could be used for intercellular Ca(2+) signaling in these cells. Endocrine cells also express extracellular ligand-gated ion channels, and their activation by hypothalamic and intrapituitary hormones leads to amplification of the pacemaking activity and facilitation of calcium influx and hormone release. These cells also express numerous G protein-coupled receptors, which can stimulate or silence electrical activity and action potential-dependent calcium influx and hormone release. Other members of this receptor family can activate calcium channels in the endoplasmic reticulum, leading to a cell type-specific modulation of electrical activity. This review summarizes recent findings in this field and our current understanding of the complex relationship between voltage-gated ion channels, ligand-gated ion channels, gap junction channels, and G protein-coupled receptors in pituitary cells.

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“…On the basis of our results, we suggest that N18 cells express mainly Ca V 3.2 channels (the most sensitive), whereas the T-type current of GH3 cells must be carried mainly by Ca V 3.1 channels. In fact, there is already evidence indicating a high expression of Ca V 3.1 mRNA in GH3 cells (Mudado et al, 2004). In addition, the observation from Lacinová et al (2000) showing that 5 mM amiloride only blocked 38% of the mouse Ca V 3.1 current implies a speciesdependent block for Ca V 3.1 channels, whereas the human clone studied here displays higher sensitivity to the diuretic (Table 1).…”

Section: Discussionmentioning

confidence: 50%

Block of Human Ca_V3 Channels by the Diuretic Amiloride

2012

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Previous studies in native T-type currents have suggested the existence of distinct isoforms with dissimilar pharmacology. Amiloride was the first organic blocker to selectively block the native T-type calcium channel, but the potency and mechanism of block of this drug on the three recombinant T-type calcium channels (Ca V 3.1, Ca V 3.2, and Ca V 3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca V 3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca V 3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca V 3 channels, we found that amiloride blocked the human Ca V 3 channels in a concentration-response manner; the IC 50 for Ca V 3.2 channels (62 M) was 13-fold lower than that for Ca V 3.1 and Ca V 3.3. Block is voltage-independent (except for Ca V 3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca V 3.1 channels. In addition, amiloride block of Ca V 3.2 channels is mainly due to an extracellular effect, whereas in Ca V 3.1 and Ca V 3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca V 3 channels differentially through a mechanism involving mainly the closed state of the channel and suggest a negative allosteric interaction with at least two putative binding sites with different affinities. The preferential block of Ca V 3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.

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CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

Cited by 11 publications

References 19 publications

Regulation of CaV3.1 Channels by Glucocorticoids

Regulation of CaV3.1 Channels by Glucocorticoids

Ion Channels and Signaling in the Pituitary Gland

Block of Human Ca_V3 Channels by the Diuretic Amiloride

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CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

Cited by 11 publications

References 19 publications

Regulation of CaV3.1 Channels by Glucocorticoids

Regulation of CaV3.1 Channels by Glucocorticoids

Ion Channels and Signaling in the Pituitary Gland

Block of Human CaV3 Channels by the Diuretic Amiloride

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Product

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Block of Human Ca_V3 Channels by the Diuretic Amiloride