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Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells
1Department of Pharmacology, Université de Sherbrooke, Que, J1H 5N4, Sherbrooke, Canada. Guylain.Boulay@USherbrooke.ca
Abstract:
Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.
Insights
Calmodulin (CaM) regulates calcium (Ca2+) entry through TRPC6 channels in mammalian cells. CaM inhibitors block receptor-operated Ca2+ entry, indicating CaM
Area of Science:
- Molecular Biology
- Cell Physiology
- Ion Channel Function
Background:
- Mammalian transient receptor potential cation channel (TRPC) homologues mediate calcium (Ca2+) influx.
- Calmodulin (CaM) is known to regulate Drosophila TRP channels and interacts with mammalian TRPCs.
- TRPC6 channels are implicated in agonist-induced Ca2+ entry in nonexcitable cells.
Purpose of the Study:
- To investigate the role of calmodulin (CaM) in regulating TRPC6-dependent calcium (Ca2+) entry.
- To determine if CaM influences receptor-operated calcium (Ca2+) entry (ROCE) mediated by TRPC6.
Main Methods:
- Stable transfection of TRPC6 into HEK-293 cells.
- Application of CaM inhibitors (calmidazolium, trifluoperazine) to assess effects on Ca2+ entry.
- Investigation of Ca2+-CaM binding to TRPC6.
Main Results:
- CaM inhibitors abolished receptor-operated Ca2+ entry (ROCE) but not thapsigargin-operated Ca2+ entry.
- CaM activity was dependent on Ca2+ complexation.
- Ca2+-CaM was shown to bind to TRPC6, and this binding was inhibited by CaM inhibitors.
Conclusions:
- Calmodulin (CaM) plays a crucial role in modulating receptor-operated calcium (Ca2+) entry (ROCE) via TRPC6 channels.
- Ca2+-bound CaM directly interacts with TRPC6, influencing its activity.
- These findings highlight a novel regulatory mechanism for TRPC6 channel function.