L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells.

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.

Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits.

Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes [Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377], the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.

Endogenous calcium channels in human embryonic kidney (HEK293) cells.

1. We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr-HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct. 2. ISr-HEK activated on depolarization to voltages positive of -40 mV. It had transient current kinetics with a time to peak of 16 +/- 1.4 ms (n = 7) and an inactivation times constant of 52 +/- 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was -19.0 +/- 1.5 mV (n = 7) and the voltage for half maximal steady-state inactivation was -39.7 +/- 2.3 mV (n = 7). 3. Block of ISr-HEK by the dihydropyridine isradipine was not stereoselective; 1 microM (+) and (-)-isradipine inhibited the current by 30 +/- 4% (n = 3) and 29 +/- 2% (n = 4) respectively. (+)-Isradipine and (-)-isradipine (10 microM) inhibited ISr-HEK by 89 +/- 4% (n = 5) and 88 +/- 8% (n = 3) respectively. The 7-bromo substituted (+/-)-isradipine (VO2605, 10 microM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83 +/- 9%, n = 3) as was observed for 10 microM (-)-nimodipine (78 +/- 6%, n = 5). Interestingly, 10 microM (+/-)-Bay K 8644 (n = 5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins omega-CTx GVIA (1 microM, inhibition by 17 +/- 3%, n = 4) and omega-CTx MVIIC (1 microM, inhibition by 20 +/- 3%, n = 4). The funnel web spider toxin omega-Aga IVA (200 nM) inhibited ISr-HEK by 19 +/- 2%, n = 4). 4. In cells expressing ISr-HEK, maximum inward current densities of 0.24 +/- 0.03 pA/pF and 0.39 +/- 0.7 pA/ pF (at a test potential of -10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 +/- 0.18 pA/pF or 1.11 +/- 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum-free medium. 5. Co-expression of a chimeric L-type calcium channel construct revealed that ISr-HEK and L-type calcium channel currents could be distinguished by their different voltage-dependencies and current kinetics. The current density after heterologous expression of the L-type alpha 1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 +/- 0.45 pA/pF) than that of ISr-HEK under the same conditions.

Calcium channels: the beta-subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the alpha 1-subunit.

A Ca2+ channel alpha 1-subunit derived from rabbit heart was transiently expressed in COS-7 cells. The dihydropyridine (+)-isradipine had low affinity (Ki = 34.3 nM) for the alpha 1-subunit in the absence of the beta-subunit due to rapid dissociation (k-1 = 0.11 min-1). Co-expression of the beta-subunit resulted in a > 35-fold increase in (+)-isradipine binding affinity (Ki = 0.9 nM) due to decreased dissociation (k-1 of 0.007 min-1). Higher DHP binding affinity was associated with an increase of the apparent affinity of Ca2+ ions for the channel. Our data suggest that the beta-subunit affects the coordination of Ca2+ ions with sites that are coupled to the dihydropyridine binding domain and by this mechanism increases the affinity for these ligands.