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

• Jeong S
• Ko J
• Kim M
• Park KC
• Park EY
• Kim J
• Baik Y
• Wie J
• Cho AE
• Jeon JH
• So I

• KMID: 2443607
• DOI: http://doi.org/10.4196/kjpp.2019.23.3.191

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.

Reference

1. Kim MJ, Jeon JP, Kim HJ, Kim BJ, Lee YM, Choe H, Jeon JH, Kim SJ, So I. Molecular determinant of sensing extracellular pH in classical transient receptor potential channel 5. Biochem Biophys Res Commun. 2008; 365:239–245.

2. Hong C, Kwak M, Myeong J, Ha K, Wie J, Jeon JH, So I. Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity. Pflugers Arch. 2015; 467:703–712.

3. Jeon JP, Roh SE, Wie J, Kim J, Kim H, Lee KP, Yang D, Jeon JH, Cho NH, Kim IG, Kang DE, Kim HJ, So I. Activation of TRPC4β by Gα_i subunit increases Ca²⁺ selectivity and controls neurite morphogenesis in cultured hippocampal neuron. Cell Calcium. 2013; 54:307–319.

4. Venkatachalam K, Zheng F, Gill DL. Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem. 2003; 278:29031–29040.

5. Sukumar P, Beech DJ. Stimulation of TRPC5 cationic channels by low micromolar concentrations of lead ions (Pb²⁺). Biochem Biophys Res Commun. 2010; 393:50–54.

6. Hong C, Seo H, Kwak M, Jeon J, Jang J, Jeong EM, Myeong J, Hwang YJ, Ha K, Kang MJ, Lee KP, Yi EC, Kim IG, Jeon JH, Ryu H, So I. Increased TRPC5 glutathionylation contributes to striatal neuron loss in Huntington's disease. Brain. 2015; 138:3030–3047.

7. Yoshida T, Inoue R, Morii T, Takahashi N, Yamamoto S, Hara Y, Tominaga M, Shimizu S, Sato Y, Mori Y. Nitric oxide activates TRP channels by cysteine S-nitrosylation. Nat Chem Biol. 2006; 2:596–607.

8. Zhu MH, Chae M, Kim HJ, Lee YM, Kim MJ, Jin NG, Yang DK, So I, Kim KW. Desensitization of canonical transient receptor potential channel 5 by protein kinase C. Am J Physiol Cell Physiol. 2005; 289:C591–C600.

9. Akbulut Y, Gaunt HJ, Muraki K, Ludlow MJ, Amer MS, Bruns A, Vasudev NS, Radtke L, Willot M, Hahn S, Seitz T, Ziegler S, Christmann M, Beech DJ, Waldmann H. (-)-Englerin A is a potent and selective activator of TRPC4 and TRPC5 calcium channels. Angew Chem Int Ed Engl. 2015; 54:3787–3791.

10. Beck A, Speicher T, Stoerger C, Sell T, Dettmer V, Jusoh SA, Abdulmughni A, Cavalié A, Philipp SE, Zhu MX, Helms V, Wissenbach U, Flockerzi V. Conserved gating elements in TRPC4 and TRPC5 channels. J Biol Chem. 2013; 288:19471–19483.

11. Ko J, Myeong J, Yang D, So I. Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s. Korean J Physiol Pharmacol. 2017; 21:133–140.

12. Rodrigues T, Sieglitz F, Somovilla VJ, Cal PM, Galione A, Corzana F, Bernardes GJ. Unveiling (-)-englerin A as a modulator of L-type calcium channels. Angew Chem Int Ed Engl. 2016; 55:11077–11081.

13. Okada T, Shimizu S, Wakamori M, Maeda A, Kurosaki T, Takada N, Imoto K, Mori Y. Molecular cloning and functional characterization of a novel receptor-activated TRP Ca²⁺ channel from mouse brain. J Biol Chem. 1998; 273:10279–10287.

14. Jung S, Mühle A, Schaefer M, Strotmann R, Schultz G, Plant TD. Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth. J Biol Chem. 2003; 278:3562–3571.

15. Gross SA, Guzmán GA, Wissenbach U, Philipp SE, Zhu MX, Bruns D, Cavalié A. TRPC5 is a Ca²⁺-activated channel functionally coupled to Ca²⁺-selective ion channels. J Biol Chem. 2009; 284:34423–34432.

16. Philipp S, Cavalié A, Freichel M, Wissenbach U, Zimmer S, Trost C, Marquart A, Murakami M, Flockerzi V. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J. 1996; 15:6166–6171.

17. Gees M, Colsoul B, Nilius B. The role of transient receptor potential cation channels in Ca²⁺ signaling. Cold Spring Harb Perspect Biol. 2010; 2:a003962.

18. Ludlow MJ, Gaunt HJ, Rubaiy HN, Musialowski KE, Blythe NM, Vasudev NS, Muraki K, Beech DJ. (-)-Englerin A-evoked cytotoxicity is mediated by Na⁺ influx and counteracted by Na⁺/K⁺-ATPase. J Biol Chem. 2017; 292:723–731.

19. Muraki K, Ohnishi K, Takezawa A, Suzuki H, Hatano N, Muraki Y, Hamzah N, Foster R, Waldmann H, Nussbaumer P, Christmann M, Bon RS, Beech DJ. Na⁺ entry through heteromeric TRPC4/C1 channels mediates (-)Englerin A-induced cytotoxicity in synovial sarcoma cells. Sci Rep. 2017; 7:16988.

20. Duan J, Li J, Zeng B, Chen GL, Peng X, Zhang Y, Wang J, Clapham DE, Li Z, Zhang J. Structure of the mouse TRPC4 ion channel. Nat Commun. 2018; 9:3102.

21. Vinayagam D, Mager T, Apelbaum A, Bothe A, Merino F, Hofnagel O, Gatsogiannis C, Raunser S. Electron cryo-microscopy structure of the canonical TRPC4 ion channel. Elife. 2008; 7:e36615.

22. Semtner M, Schaefer M, Pinkenburg O, Plant TD. Potentiation of TRPC5 by protons. J Biol Chem. 2007; 282:33868–33878.

23. Carson C, Raman P, Tullai J, Xu L, Henault M, Thomas E, Yeola S, Lao J, McPate M, Verkuyl JM, Marsh G, Sarber J, Amaral A, Bailey S, Lubicka D, Pham H, Miranda N, Ding J, Tang HM, Ju H, et al. Englerin A agonizes the TRPC4/C5 cation channels to inhibit tumor cell line proliferation. PLoS One. 2015; 10:e0127498.

24. Rubaiy HN, Seitz T, Hahn S, Choidas A, Habenberger P, Klebl B, Dinkel K, Nussbaumer P, Waldmann H, Christmann M, Beech DJ. Identification of an (-)-englerin A analogue, which antagonizes (-)-englerin A at TRPC1/4/5 channels. Br J Pharmacol. 2018; 175:830–839.

25. Chen X, Li W, Riley AM, Soliman M, Chakraborty S, Stamatkin CW, Obukhov AG. Molecular determinants of the sensitivity to G_q/11-phospholipase C-dependent gating, Gd³⁺ potentiation, and Ca²⁺ permeability in the transient receptor potential canonical type 5 (TRPC5) channel. J Biol Chem. 2017; 292:898–911.

26. Duan J, Li J, Chen GL, Xie K, Peng X, Zhou W, Zhong J, Zhang Y, Xu J, Xue C, Zhu L, Liu W, Tian XL, Wang J, Zeng B, Clapham DE, Li Z, Zhang J. Cryo-EM structure of the receptor-activated TRPC5 ion channel at 29 angstrom resolution. bioRxiv. 2018; 2018:467969.

27. Li J, Zhang X, Song X, Liu R, Zhang J, Li Z. The structure of TRPC ion channels. Cell Calcium. 2019; 80:25–28.

28. Kim H, Kim J, Jeon JP, Myeong J, Wie J, Hong C, Kim HJ, Jeon JH, So I. The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels. Channels (Austin). 2012; 6:333–343.