Related Experiment Videos
cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels
G Mikala1, U Klöckner, M Varadi
1Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, OH 45267-0828, USA.
Abstract:
The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.
Insights
This study investigated cardiac L-type Ca2+ channels and found that protein kinase A (PKA) phosphorylation sites do not affect their basal activity or dihydropyridine binding. Deleting parts of the channel increased expression and current, but PKA modulation remained ineffective.
Area of Science:
- Molecular Biology
- Cardiovascular Physiology
- Ion Channel Function
Background:
- Cardiac L-type Ca2+ channels are crucial for heart function.
- Their activity is regulated by various signaling pathways, including cAMP-dependent phosphorylation.
- The precise role of specific phosphorylation sites in basal channel activity is not fully understood.
Purpose of the Study:
- To investigate the involvement of cAMP-dependent phosphorylation sites in the basal activity of cardiac L-type Ca2+ channels.
- To determine the effect of eliminating consensus and nonconsensus protein kinase A (PKA) sites on channel function.
- To assess the impact of PKA modulation on channel activity in cells expressing wild-type and mutant channels.
Main Methods:
- HEK 293 cells were transiently cotransfected with mutants of human cardiac alpha1 and accessory subunits of L-type Ca2+ channels.
- Systematic elimination of high consensus PKA sites was achieved via serine to alanine substitutions.
- Deletion mutants were created to explore nonconsensus sites, and carboxyl-terminal truncations were analyzed.
- Dihydropyridine (DHP) binding assays were performed.
- PKA activity was modulated to assess its effect on channel function.
Main Results:
- Elimination of high consensus PKA sites did not alter Ca2+ channel currents or dihydropyridine binding characteristics.
- Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 led to increased channel expression and current amplitudes.
- Modulation of PKA activity did not affect Ca2+ channel functions in cells expressing wild-type or mutant channels.
Conclusions:
- Consensus PKA phosphorylation sites are not essential for establishing basal activity or DHP binding of cardiac L-type Ca2+ channels in this expression system.
- Nonconsensus sites or other regulatory mechanisms may play a role in channel expression and function.
- Cardiac Ca2+ channels expressed in HEK 293 cells exhibit a lack of PKA control, similar to smooth muscle cell channels.