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Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase
L H Jiang1, D J Gawler, N Hodson
1School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Abstract:
We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.
Insights
8-bromo-cyclic GMP (8-Br-cGMP) inhibits cardiac L-type calcium channel currents by activating protein kinase G. This inhibition occurs through phosphorylation of the alpha(1C) subunit at Serine 533.
Area of Science:
- Cardiovascular Physiology
- Molecular Pharmacology
- Ion Channel Function
Background:
- L-type calcium channels are crucial for cardiac function.
- Cyclic GMP (cGMP) is a key second messenger involved in various cellular processes.
- The precise role of cGMP in regulating cardiac L-type calcium channels requires further elucidation.
Purpose of the Study:
- To investigate the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents.
- To determine the specific site and mechanism of action of 8-Br-cGMP on these channels.
Main Methods:
- Expression of rabbit cardiac alpha(1C) subunit, with or without beta subunits, in Xenopus oocytes.
- Two-electrode voltage-clamp recordings to measure calcium channel currents.
- Site-directed mutagenesis (Serine to Alanine substitutions) and application of protein kinase G inhibitor (KT5823).
Main Results:
- 8-Br-cGMP application decreased calcium channel currents in cells expressing the alpha(1C) subunit.
- Inhibition was blocked by the protein kinase G inhibitor KT5823.
- Mutagenesis identified Serine 533 on the alpha(1C) subunit as the critical site for 8-Br-cGMP-mediated inhibition.
Conclusions:
- cGMP inhibits cardiac L-type calcium channel currents.
- This inhibition is mediated by protein kinase G.
- Phosphorylation of the alpha(1C) subunit at Serine 533 is the key mechanism underlying cGMP's effect.