Functional Properties of CaV1.3 (α1D) L-type Ca2+ Channel Splice Variants Expressed by Rat Brain and Neuroendocrine GH3 Cells

Abstract

Ca2؉ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca 2؉ entry through activation of pertussis toxin-sensitive G proteins leading to activation of K ؉ channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca 2؉ channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three ␣ 1 (␣ 1A , ␣ 1C , and ␣ 1D ), four ␤, and one ␣ 2 -␦ subunit in rat pituitary GH 3 cells; ␣ 1B and ␣ 1S transcripts were absent. GH 3 cells express multiple alternatively spliced ␣ 1D mRNAs. Many of the ␣ 1D transcript variants encode "short" ␣ 1D (␣ 1D-S ) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other ␣ 1 subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (␣ 1D-L ). We cloned and expressed the predominant ␣ 1D-S transcript variants in rat brain and GH 3 cells and their ␣ lD-L counterpart in GH 3 cells. Unlike ␣ 1A channels, ␣ 1D channels exhibited current-voltage relationships similar to those of native GH 3 cell Ca 2؉ channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced ␣ 1D transcripts form functional Ca 2؉ channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of ␣ 1D-S subunit, is not sufficient to confer sensitivity to inhibitory G proteins.

Erbin Enhances Voltage-Dependent Facilitation of Cav1.3 Ca2+Channels through Relief of an Autoinhibitory Domain in the Cav1.3 α1Subunit

“…5A). Consistent with previous studies (Safa et al, 2001), a short (20 ms) conditioning prepulse from Ϫ90 to ϩ60 mV caused a significant increase (30.3 Ϯ 4.1%; n ϭ 12; p Ͻ 0.01) in Ca v 1.3 currents evoked by a test pulse from Ϫ90 to Ϫ20 mV (Fig. 5A).…”

“…Ca v 1.3 channels undergo prominent VDF that, in recombinant systems, is independent of heterotrimeric G-proteins or phosphorylation (Safa et al, 2001). To determine whether erbin influenced this aspect of Ca v 1.3 function, we used a paired-pulse protocol in which the amplitude of two test currents (P1 and P2) evoked by the same voltage step was compared before and after a conditioning (facilitating) prepulse (Fig.…”

“…To characterize the importance of the PDZ-binding site in the ␣ 1 1.3 CT for erbin modulation, we took advantage of a naturally occurring ␣ 1 1.3 variant (␣ 1 1.3b) in which inclusion of an alternatively spliced exon leads to truncation of the C-terminal domain and subsequent elimination of the PDZ-binding sequence (Safa et al, 2001;Xu and Lipscombe, 2001) (Fig. 8 A).…”

Effects of low-dose ionising radiation on pituitary adenoma: is there a role for L-type calcium channel?

“…SOCE is part of the calcium influx stimulated by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells and regulates hypophyseal secretion of prolactin (Villalobos & Garcia-Sancho, 1995). GH3 presents other calcium channels than Ca v 1 (Lomeo et al, 2004), such as Ca v 2 (Safa et al, 2001) and store-operated Ca 2+ channels (Villalobos & Garcia-Sancho, 1995). The results obtained in this study argue against the involvement of Cav1 in the low dose ionising radiation effect on GH3 cells.…”

Interactions with PDZ Proteins Are Required for L-Type Calcium Channels to Activate cAMP Response Element-Binding Protein-Dependent Gene Expression

“…ITTL are the last four amino acids of one splice variant of the calcium channel ␣ 1D (Ca V 1.3) subunit (Ihara et al, 1995;Safa et al, 2001) and were not predicted to interact with PDZ binding domains. As with EGFP, EGFP-ITTL exhibited a homogenous distribution throughout the cell, without altering ␣ 1C labeling (Fig.…”