Isoform- and receptor-specific channel property of canonical transient receptor potential (TRPC)1/4 channels (original) (raw)

Abstract

Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1β) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1β/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a β ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1β/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.

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Abbreviations

TRPC:

Transient receptor potential canonical

TRPC1α:

human TRPC1 isoform long

TRPC1β:

human TRPC1 isoform short

CCh:

(2-Hydroxyethyl)trimethylammonium chloride carbamate

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Acknowledgements

We thank Dr. Michael Mederos and Dr. Thomas Gudermann for kindly donating human TRPC1 isoform long (hTRPC1α) construct and Dr. Vaca Luis for kindly donating CFP-hTRPC1 isoform long and YFP-hTRPC1 isoform long constructs. We thank Dr. Young Duk Yang for kindly donating rat histamine receptor type 1. We thank Dr. Yin Hua Zhang for kindly revising the manuscript. We acknowledge the roles of the Biomedical Imaging Center at the Seoul National University College of Medicine, Seoul, Korea. This study was supported by grant from the National Research Foundation of Korea funded by the Korea government (MEST) (2012R1A2A1A01003073) and Seoul National University Hospital Research Fund (03-2012-0140) (I. So).

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea
    Jinsung Kim
  2. Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Republic of Korea
    Jinsung Kim, Misun Kwak, Jae-Pyo Jeon, Jongyun Myeong, Jinhong Wie, Chansik Hong, Sung-Young Kim, Ju-Hong Jeon & Insuk So
  3. Department of Physiology, Sungkyunkwan University School of Medicine, Suwon, 440-746, Republic of Korea
    Hyun Jin Kim

Authors

  1. Jinsung Kim
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  2. Misun Kwak
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  3. Jae-Pyo Jeon
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  4. Jongyun Myeong
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  5. Jinhong Wie
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  6. Chansik Hong
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  7. Sung-Young Kim
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  8. Ju-Hong Jeon
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  9. Hyun Jin Kim
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  10. Insuk So
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Corresponding authors

Correspondence toHyun Jin Kim or Insuk So.

Additional information

Jinsung Kim and Misun Kwak contributed equally to this work and should be considered as co-first authors.

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Supplementary Fig. 1

Surface level of TRPC1α, TRPC4β channels in their homo/heteromeric formation. (a) Surface biotinylation for HEK293 cells expressing TRPC1α, TRPC4β, and TRPC1α+TRPC4β showed that TRPC1α has significantly lower surface level compared to TRPC4β channels. TRPC1α channel translocates into plasma membrane when TRPC4β is co-expressed (PPTX 122 kb)

Supplementary Fig. 2

Currents measured in HEK293 cells expressing TRPC1α/5, TRPC1αLFW/AAA/5 channels. (a, b) Left panel indicate I_–_V curves and right panel indicate corresponding current traces. In response to extracellular carbachol treatment (CCh, 100 μM), cells expressing TRPC1α and TRPC5 showed typical double-rectifying I_–_V curves. (c), (d) Left panel indicate I_–_V curves and right panel indicate corresponding current traces. In response to extracellular carbachol treatment (CCh, 100 μM), cells expressing TRPC1αLFW/AAA and TRPC5 showed double-rectifying I_–_V curves (PPTX 857 kb)

Supplementary Fig. 3

Cation conductance curves of outward-rectifying TRPC1α/4 channels and double-rectifying TRPC4 channels. (a) Left panel shows representative outward-rectifying I_–_-V curve of TRPC1α/4 channel and right panel shows representative double-rectifying I_–_V curve of TRPC4 channel. (b) Left panel shows normalized conductance (G)–-voltage (V) plot of TRPC1α/4 channels and right panel shows normalized G_–_V plot of TRPC4 channels. (c) Left panel indicates G–V plots shown in (b) and their fitting result to Boltzmann equation. Cation conductance was obtained by dividing current amplitude at each potential with driving force at that membrane potential. Four vertical dotted lines are drawn at voltage points of −120, −80, 10, and 50 mV. Light gray color represents typical N- shaped G_–_V plot of TRPC4 channels; gray color represents monotonically increasing G_–_V plot of TRPC1α/4 channels; black lines represent curve fitting results to Boltzmann equation. Right panel summarizes some of the parameters that characterize each Boltzmann equations used for curve fitting and thereby each cation conductance curves (PPTX 236 kb)

Supplementary Fig. 4

Activity of TRPC4 channel in response to serial application of carbachol and histamine. (a–c) Carbachol-M2R-activated TRPC4 channel allow only a minute additional activation to histamine (n = 4, 4.07 ± 1.53 %). (PPTX 309 kb)

Supplementary Fig. 5

TRPC1 lacks one of the TRPC4-Gαi2 binding interfaces: K715R716. (a) Amino acid alignment of human TRPC1 isoform long, human TRPC1 isoform short, mouse TRPC4 isoform long, mouse TRPC4 isoform short, and mouse TRPC5. The binding interface between TRPC4 and Gαi2 protein is characterized by signature sequence RNXXKR as highlighted by red square. Although this site is totally conserved in TRPC4α, TRPC4β, and TRPC5, TRPC1 has CCXXHR instead of RNXXKR. Two cysteine motif and H-switched K might obligatory for M2R selectivity. (PPTX 1170 kb)

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Kim, J., Kwak, M., Jeon, JP. et al. Isoform- and receptor-specific channel property of canonical transient receptor potential (TRPC)1/4 channels.Pflugers Arch - Eur J Physiol 466, 491–504 (2014). https://doi.org/10.1007/s00424-013-1332-y

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