Mapping of the pore structure of the vanilloid receptor channel TRPV1

k. susankova¹, J. Teisinger¹, L. Vyklicky¹, F. Viana², R. Ettrich³ and V. Vlachova¹

¹Institute of Physiology, Academy of Sciences, 142 20 Prague 4, Czech Republic,

²Institute of Neuroscience of Alicante, UMH-CSIC, San Juan de Alicante, Spain,

³Laboratory of High Performance Computing, Institute of Physical Biology USB and Institute of Landscape Ecology AS CR, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

The vanilloid receptor (TRPV1) is a nonselective cation channel that is predominantly expressed by nociceptive sensory neurons. This channel is activated by a wide array of pain-producing stimuli, including capsaicin, protons and noxious heat (> 43°C) [1]. Similar to other members of the transient receptor potential (TRP) family, the structure of the TRPV1 represents six putative transmembrane segments S1-S6, a pore region (P-loop) connecting S5 and S6, and cytoplasmic hydrophilic N- and C-terminal regions. The functional TRPV1 channel is assembled from four identical subunits. The central ion conducting pore domain is supposed to be formed by S5-S6 and the P-loop region. Structure-function studies have recently identified several key domains that contribute to the activation and the modulation of the TRPV1 receptor. However, little is known about the structural rearrangements that lead to the channel gating and ion conduction.

To obtain an insight into the pore architecture of TRPV1 and to elucidate how this region affects the ion selectivity and permeation properties of this channel, we subjected the sequence from E570 to E694 of the rat TRPV1 receptor to homology modeling. This part includes S5 and S6 helices and a loop region containing one inner helix. Based upon the predicted structure and the alignment of the pore region with related channels TRPV5, TRPV6 and KcsA [2,3], we selected three amino acids for site-directed PCR mutagenesis in order to characterize their role in channel gating and ion permeation. As the first step of the prospective cystein-scanning mutagenesis, we substituted glycine for all endogenous cysteines that are putatively exposed to the extracellular milieu. This construct was functional [4] and served as a template for the introduction of three individual cysteine substitution mutants M644C, D646C, and E648C.

HEK293T cells transiently expressing either the wild type or the respective mutant receptor were assayed by patch-clamp technique and calcium imaging. Mutation of M644C strongly reduced the magnitude of the heat induced responses, whereas the capsaicin sensitivity remained unaltered as compared with wild type. A similar pattern of responsiveness has been observed in a mutant channel in which M644 was replaced by alanine. This mutant was less permeable to Ca²⁺ exhibiting a decrease in ratio of Ca²⁺ to Na⁺ permeability from 5.0 ± 2.4 to 1.6 ± 0.2 (n = 4 and 10) for capsaicin activation and from 4.2 ± 1.1 to 1.7 ± 0.2 (n = 4 and 9) for heat stimulation. The D646C mutant appeared to be nonfunctional. Mutation of E648C rendered the channel insensitive to both thermal and chemical stimuli when applied individually; however, heat in combination with capsaicin elicited robust responses in calcium imaging experiments.

Data from this study substantiate the proposed pore organization of the TRPV1 channel and suggest that the pore-lining residues M644, D646 and E648 are important molecular determinants that govern key properties of ion permeation and channel gating.

This work was supported by Grants 305/03/0802 and 309/04/0496 of the Grant Agency of the Czech Republic, by Grant 2003CZ0020 of CSIC, Research Project of the AS CR, AVOZ 50110509 and 60870520 and by the Ministry of Education, Youth and Sports of the Czech Republic, 1M0002375201 and MSM6007665808

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