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Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids

  • Ion channels, receptors and transporters
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An Erratum to this article was published on 10 May 2016

Abstract

Transient receptor potential canonical (TRPC) 4 channels are calcium-permeable, nonselective cation channels and are widely expressed in mammalian tissue, especially in the GI tract and brain. TRPC4 channels are known to be involved in neurogenic contraction of ileal smooth muscle cells via generating cationic current after muscarinic stimulation (muscarinic cationic current (mIcat)). Polyamines exist in numerous tissues and are believed to be involved in cell proliferation, differentiation, scar formation, wound healing, and carcinogenesis. Besides, physiological polyamines are essential to maintain inward rectification of cardiac potassium channels (Kir2.1). At membrane potentials more positive than equilibrium potential, intracellular polyamines plug the cytosolic surface of the Kir2.1 so that potassium ions cannot pass through the pore. Recently, it was reported that polyamines inhibit not only cardiac potassium channels but also nonselective cation channels that mediate the generation of mIcat. Here, we report that TRPC4, a definite mIcat mediator, is inhibited by intracellular spermine with great extent. The inhibition was specific to TRPC4 and TRPC5 channels but was not effective to TRPC1/4, TRPC1/5, and TRPC3 channels. For this inhibition to occur, we found that glutamates at 728th and 729th position of TRPC4 channels are essential whereby we conclude that spermine blocks the TRPC4 channel with electrostatic interaction between negative amino acids at the C-terminus of the channel.

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Acknowledgments

We thank Dr. Michael Mederos and Dr. Thomas Gudermann for kindly donating human TRPC1 isoform long (hTRPC1α) construct. TRPC5 were kindly donated by Dr S. Kaneko and Dr Y. Mori. TRPC4 were kindly donated by Dr V. Flockerzi and Dr. M. Schaefer.

This study was supported by grants from the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology) and Future Planning (MSIP) of the Korean Government (MSIP) (2013R1A1A1010783) (K.P. Lee), (2012R1A2A1A01003073) (I. So). Y. S. was supported by the BK21 plus program from the MSIP. This work was supported by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So).

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    Authors and Affiliations

    1. College of Medicine, Catholic University of Korea, Seoul, 137-701, Republic of Korea

      Jinsung Kim

    2. Department of Physiology, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea

      Jinsung Kim, Young-Cheul Shin, Ju-Hong Jeon & Insuk So

    3. Department of Surgery, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea

      Sang Hui Moon & Kyu Joo Park

    4. Department of Physiology, College of Veterinary Medicine, Chungnam National University, Daejeon, 305-764, Republic of Korea

      Kyu Pil Lee

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    Jinsung Kim and Sang Hui Moon contributed equally to this work.

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    Supplementary Figure 1

    Effect of intracellular spermidine and putrescine to TRPC4 channel. (a), (b) all left panels indicate current traces and right panels indicate corresponding I-V curves at noted points (1, 2 and 3). 1 mM of spermidine and putrescine could not inhibit TRPC4 channel as strong as spermine. There was no asymmetry between outward current and inward current as was in spermine. Both polyamines, however, somewhat slowed down the activation process. (PPTX 202 kb)

    Supplementary Figure 2

    Intracellular spermine inhibit TRPC3 with nonspecific manner. (a) Normalized current density of TRPC3 channels. White circle indicates currents without spermine and black circle indicates current with intracellular spermine. Two currents showed indifferent I-V shape. (b) Current density of TRPC3 current with and without intracellular spermine. Intracellular spermine reduced both inward and outward current density of TRPC3 channels. (c) Current trace for TRPC3 recording. Muscarinic acetylcholine receptor 3 and extracellular carbachol (CCh, 100 μM) was used for activation of TRPC3 channels. In addition, divalent-free solution (DVF) was used in the activation process. (PPTX 106 kb)

    Supplementary Figure 3

    Distribution of negative amino acids in C-terminus of TRPC4 channel. (a) Amino acid sequence of C-terminus of TRPC4 channel. Red capitals indicate negative amino acids (D and E), green capitals indicate TRP box sequence (EWKFAR) and gray capitals indicate alternative splicing site for TRPC4β. (b) Left panel, From 621st amino acid to 890th amino acid in TRPC4β, negative amino acids showed sporadic distribution when window width was set to 20 amino acids. When 740th amino acid was set as cut-off number, however, distribution of negative amino acids showed clear stratification. (PPTX 65 kb)

    Supplementary Figure 4

    Effect of high magnesium ([Mg2+]i = 10 mM) on TRPC4 channels. (a) I-V curves for whole-cell TRPC4 current with 10 mM of intracellular Mg2+. Augmented intracellular Mg2+ could not alter the shape of I-V curves. (b) Representative current race of TRPC4 current under 10 mM intracellular Mg2+. Activation kinetics were similar to physiological condition ([Mg2+]i ∼ 3 mM). (PPTX 390 kb)

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    Kim, J., Moon, S.H., Shin, YC. et al. Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids. Pflugers Arch - Eur J Physiol 468, 551–561 (2016). https://doi.org/10.1007/s00424-015-1753-x

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