Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation
10.4196/kjpp.2019.23.3.191
- Author:
SeungJoo JEONG
1
;
Juyeon KO
;
Minji KIM
;
Ki Chul PARK
;
Eunice Yon June PARK
;
Jinsung KIM
;
Youngjoo BAIK
;
Jinhong WIE
;
Art E CHO
;
Ju hong JEON
;
Insuk SO
Author Information
1. Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea. Insuk@snu.ac.kr
- Publication Type:Original Article
- Keywords:
Englerin A;
Ion channels;
Mutant proteins;
Transient receptor potential canonical 5
- MeSH:
Calcium;
Carbachol;
Humans;
Hydrogen-Ion Concentration;
Ion Channels;
Kidney;
Mass Screening;
Mutagenesis, Site-Directed;
Mutant Proteins;
Protein Kinase C;
Receptors, Muscarinic
- From:The Korean Journal of Physiology and Pharmacology
2019;23(3):191-201
- CountryRepublic of Korea
- Language:English
-
Abstract:
The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.
