A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart

Haase, Hannelore; Striessnig, Jörg; Holtzhauer, Martin; Vetter, Roland; Glossmann, Hartmut

doi:10.1016/s0922-4106(05)80037-9

Cited by 33 publications

(27 citation statements)

References 25 publications

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“…1,2,3). This result is in close agreement with tindings previously obtained by us [9,17] with calcium channels purified from porcine heart as well as by Yoshida et al [20] who used an anti-a, antibody to precipitate phosphorylated rabbit cardiac calcium channels. But, in contrast to the latter authors we did not detect the phosphorylated 250 kDa full-length form of the ~1, subunit.…”

Section: Other Assayssupporting

confidence: 92%

“…Biochemical studies using purified calcium channels gave no indication for a CAMP-dependent phosphoryiation of the a, subunit [8,9,17]. This is in line [9]. The catalytic subunit of PKA was prepared from bovine heart according to [25].…”

Section: I~rodu~onmentioning

confidence: 99%

“…Insoluble material was removed by centrifugation at 100,000 x g for 30 min. The DHP receptor of the canine cardiac calcium channel was prelabeled with 10 nM (+)-[3H]PN20(rl10 as described [9]. The ryanodine receptor of the calcium release channel was prelabeled with 5 nM of [3H]ryanodine according to [31].…”

Section: Phosphorylation and Labeling Of Receptors In Cardiac Membranesmentioning

confidence: 99%

“…Following a binding and washing procedure, the radioactivity bound to the beads was measured by liquid scintillation counting. The amount of receptor sites remaining in the supematant was determined by polyethylene glycol precipitation [9]. All procedures were performed under yellow light.…”

Section: Immunoprecipitationmentioning

confidence: 99%

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Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

et al. 1993

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Cyclic AMP-rnediat~ phospho~la~on of calcium channel subunits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by CAMP-dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy-terminal sequence of the cardiac /I subunit. A 62 kDa protein was identified as the major PKA-substrate in the immunoprecipitates. In the intact myocardium, this putative p subunit was found to be phosphorylated in response to CAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the a, subunit was detected, although 1,4diiydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that IX&mediated phospho~lation of the b subunit is the major mechanism for @-adrenergic regulation of cardiac L-type calcium channel activity.

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Section: Other Assayssupporting

confidence: 92%

Section: I~rodu~onmentioning

confidence: 99%

Section: Phosphorylation and Labeling Of Receptors In Cardiac Membranesmentioning

confidence: 99%

Section: Immunoprecipitationmentioning

confidence: 99%

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Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

et al. 1993

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“…COS-7 cells (ATCC CRL 165i) were transfected following a modified DEAE-Dextran protocol [17] weeks at -SOT until use. Guinea-pig cardiac membranes were preseparated by filtration over GF/C glass fibre filters using 10% (w/v) pared as described [18]. Binding assays were carried out in 50 mM polyethylene glycol 6000, 10 mM Tris-HCI, pH 7.4, 10 mM MgCI, as Tris-HCI (pH 7.4 (25"C)), 0.1 mM PMSF, 1 mM CaCl,, 1 mg/ml bovine the washing buffer.…”

Section: Construction Of Expression Plasmids and Transfectionmentioning

confidence: 99%

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca²⁺ binding sites of the α₁‐subunit

et al. 1994

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

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Expression of Ca2+ channel subunits during cardiac ontogeny in mice and rats: Identification of fetal ?1C and ? subunit isoforms

et al. 2000

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Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.

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A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart

Cited by 33 publications

References 25 publications

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca²⁺ binding sites of the α₁‐subunit

Expression of Ca2+ channel subunits during cardiac ontogeny in mice and rats: Identification of fetal ?1C and ? subunit isoforms

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A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart

Cited by 33 publications

References 25 publications

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the α1‐subunit

Expression of Ca2+ channel subunits during cardiac ontogeny in mice and rats: Identification of fetal ?1C and ? subunit isoforms

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Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca²⁺ binding sites of the α₁‐subunit