Korean J Physiol Pharmacol.  2019 May;23(3):191-201. 10.4196/kjpp.2019.23.3.191.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

Affiliations
  • 1Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea. Insuk@snu.ac.kr
  • 2College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea.
  • 3Department of Bioinformatics, Korea University, Sejong 30019, Korea.
  • 4Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.

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.

Keyword

Englerin A; Ion channels; Mutant proteins; Transient receptor potential canonical 5

MeSH Terms

Calcium
Carbachol
Humans
Hydrogen-Ion Concentration
Ion Channels
Kidney
Mass Screening
Mutagenesis, Site-Directed
Mutant Proteins
Protein Kinase C
Receptors, Muscarinic
Calcium
Carbachol
Ion Channels
Mutant Proteins
Protein Kinase C
Receptors, Muscarinic

Figure

  • Fig. 1 Englerin A (EA)-induced currents were abolished in TRPC5K554N, TRPC5H594N, and TRPC5E598Q. (A) A representative full current trace of TRPC5WT induced by 100 nM of EA (top) and the current (I)-voltage (V) relationship of current increases (b−a) by EA (bottom). The a, b indicate the basal current (a) and the EA-induced peak current (b). The currents were recorded in TRPC5-expressing HEK293 cells using the whole-cell patch clamp technique. At a holding potential of −60 mV, the ramp pulse was applied from 100 mV to −100 mV at every 20 sec. (B) A representative full current trace of TRPC5K554N induced by EA (top) and the I–V relationship of current increases by EA (bottom). (C) A representative full current trace of TRPC5H594N induced by EA (top) and the I–V relationship of current increases by EA (bottom). (D) A representative full current trace of TRPC5E598Q induced by EA (top) and the I–V relationship of current changes by EA (bottom). (E) A summarized current density at −60 mV of TRPC5WT and mutants stimulated by EA (100 nM). TRPC5, transient receptor potential canonical 5; WT, wild type; HEK, human embryonic kidney. *p < 0.05 and **p < 0.01. The dotted gray lines in (A–D) indicate baseline.

  • Fig. 2 Effects of englerin A on TRPC5 mutants. (A) A representative full current trace (top) and the I–V relationship of current increases (b−a) by EA (bottom) of TRPC5R545N (A-a), TRPC5E549Q (A-b), TRPC5K560N (A-c), and TRPC5E570Q (A-d). (B) A representative full current trace (top) and the I–V relationship of current increases by EA (bottom) of TRPC5E543Q (B-a), TRPC5R557N (B-b), TRPC5R593N (B-c), and TRPC5E595Q (B-d). The dotted gray line indicates baseline. The a, b indicate the basal current (a) and the EA-induced peak current (b). TRPC5, transient receptor potential canonical 5.

  • Fig. 3 Effects of englerin A (EA) on GTPγS-induced current increases of TRPC5WT and TRPC5 mutants. (A) A representative full current trace of TRPC5WT induced by intracellular GTPγS and EA (top) and the I–V relationship of current increases (b−a) by EA (bottom). The a, b indicates the basal current (a) and the peak current (b). (B) A representative full current trace of TRPC5H594N induced by intracellular GTPγS and EA (top) and the I–V relationship of current increases by EA (bottom). (C) A summarized current density at −60 mV of TRPC5WT, TRPC5K554N, TRPC5H594N, and TRPC5E598Q induced by intracellular GTPγS and EA under the condition of Na (open column) and under the condition of Cs+ (filled column). TRPC5, transient receptor potential canonical 5; WT, wild type; GTPγS, guanosine 5′-O-[gamma-thio]triphosphate. *p < 0.05 and **p < 0.001. The dotted gray line in A and B indicates baseline.

  • Fig. 4 Effects of carbachol (CCh) on TRPC5WT and TRPC5 mutants. (A) A summarized current density at −60 mV of TRPC5WT and the mutants with CCh stimulation under condition of Na. (B) A summarized current density at −60 mV of TRPC5WT and the mutants with CCh stimulation under condition of Na in HEK cells expressing human M3 muscarinic receptor. (C) A representative full current trace of TRPC5WT, TRPC5H594N, and TRPC5E598Q induced by CCh (top) and the I–V relationship of current increases by CCh (bottom). After establishing the whole-cell configuration, the external solution was changed from NT to Cs+-rich solution for recording TRPC5 current in HEK293 cells. The a, b, c, d indicates the basal current (a) and the peak current (b) under the condition of Cs+, and the basal current (c) and the peak current (d) under the condition of Cs+ and CCh. The I–V relationship of current increases by Cs (b−a) was obtained by subtracting a from b and the I–V relationship of current increases by CCh (d-c) by subtracting c from d. (D) A summarized current density at −60 mV of TRPC5WT and the mutants with Cs+. (E) A summarized current density at −60 mV of TRPC5WT and the mutants with CCh. TRPC5, transient receptor potential canonical 5; WT, wild type; n.s., not significant; HEK, human embryonic kidney. *p < 0.05 and **p < 0.01. The dotted gray line indicates baseline.

  • Fig. 5 Effects of 1 µM of englerin A (EA) on TRPC5WT and TRPC5 mutants. (A) A representative full current trace (left panel) and the I–V relationship of current increases (b−a) by EA (right panel) of TRPC5K554N (A-a), TRPC5H594N (A-b), and TRPC5E598Q (A-c). The a, b indicates the basal current (a) and the EA-induced peak current (b). (B) A summarized current density at −60 mV of TRPC5WT and the mutants with EA. The dotted gray line indicates baseline. TRPC5, transient receptor potential canonical 5; WT, wild type.


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