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1.
The Korean Journal of Physiology and Pharmacology ; : 191-201, 2019.
Article in English | WPRIM | ID: wpr-761784

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.


Subject(s)
Humans , Calcium , Carbachol , Hydrogen-Ion Concentration , Ion Channels , Kidney , Mass Screening , Mutagenesis, Site-Directed , Mutant Proteins , Protein Kinase C , Receptors, Muscarinic
2.
Experimental & Molecular Medicine ; : 160-169, 2007.
Article in English | WPRIM | ID: wpr-90617

ABSTRACT

In our previous study, two point mutants of apolipoprotein A-I, designated V156K and A158E, revealed peculiar characteristics in their lipid-free and lipid-bound states. In order to determine the putative therapeutic potential of these mutants, several in vitro and in vivo evaluations were conducted. In the lipid-free state, V156K showed more profound antioxidant activity against LDL oxidation than did the wildtype (WT) or A158E variants in an in vitro assay. In the lipid-bound state, V156K-rHDL showed an enhanced cholesterol delivery activity to HepG2 cells in a time-dependent manner, as compared to WT-rHDL, A158E-rHDL, and R173C-rHDL. We assessed the physiological activities of the mutants in circulation, using hypercholesterolemic mice (C57BL6/J). Palmitoyloleoyl phosphatidylcholine (POPC)-rHDL preparations containing each of the apoA-I variants were injected into the mice at a dosage of 30 mg of apoA-I/kg of body weight. Forty eight hours after injection, the sera of the V156K-rHDL injected group showed the most potent antioxidant abilities in the ferric acid removal assay. The V156K-rHDL- or R173C-rHDL-injected mice showed no atherosclerotic lesions and manifested striking increases in their serum apo-E levels, as compared to the mice injected with WT-rHDL or A158E-rHDL. In conclusion, V156K-rHDL exhibited the most pronounced antioxidant activity and anti-atherosclerotic activity, both in vitro and in vivo. These results support the notion that HDL-therapy may prove beneficial due to its capacity to induce accelerated cholesterol excretion, as well as its enhanced antioxidant and anti-inflammatory effects and lesion regression effect.


Subject(s)
Animals , Humans , Male , Mice , Amino Acids/genetics , Antioxidants/metabolism , Apolipoprotein A-I/genetics , Atherosclerosis/pathology , Biological Transport/drug effects , Cell Line, Tumor , Cholesterol/metabolism , Copper/pharmacology , Hypercholesterolemia/chemically induced , Lipoproteins, HDL/blood , Lipoproteins, LDL/blood , Mice, Inbred C57BL , Oxidation-Reduction/drug effects , Point Mutation/genetics , Recombinant Proteins/blood
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