Elsevier

Cell Calcium

Volume 84, December 2019, 102106
Cell Calcium

Structural biology of thermoTRPV channels

https://doi.org/10.1016/j.ceca.2019.102106Get rights and content

Highlights

  • Cryo-EM and X-ray structures of TRPV channels.

  • Structural basis of ion selectivity and gating.

  • Conformational landscapes of TRPV channels.

  • Channel-lipid interactions.

Abstract

Essential for physiology, transient receptor potential (TRP) channels constitute a large and diverse family of cation channels functioning as cellular sensors responding to a vast array of physical and chemical stimuli. Detailed understanding of the inner workings of TRP channels has been hampered by a lack of atomic structures, though structural biology of TRP channels has been an enthusiastic endeavor since their molecular identification two decades ago. These multi-domain integral membrane proteins, exhibiting complex polymodal gating behavior, have been a challenge for traditional X-ray crystallography, which requires formation of well-ordered protein crystals. X-ray structures remain limited to a few TRP channel proteins to date. Fortunately, recent breakthroughs in single-particle cryo-electron microscopy (cryo-EM) have enabled rapid growth of the number of TRP channel structures, providing tremendous insights into channel gating and regulation mechanisms and serving as foundations for further mechanistic investigations. This brief review focuses on recent exciting developments in structural biology of a subset of TRP channels, the calcium-permeable, non-selective and thermosensitive vanilloid subfamily of TRP channels (TRPV1-4), and the permeation and gating mechanisms revealed by structures.

Introduction

TRP channels are central to numerous biological processes by sensing a multitude of environmental cues and physiological stimuli [[1], [2], [3], [4], [5]]. Consequently, mutations of human TRP channel genes have been associated with a wide spectrum of inherited diseases in the musculoskeletal, cardiovascular, and nervous systems [6]. On the basis of sequence similarity, the mammalian TRP channel subunit genes can be divided into six subfamilies - TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV [2]. Functional TRP channels are composed of homo- or hetero- tetramers, which are architecturally related to voltage-gated ion channels (VGIC) such as voltage-gated sodium (NaV), potassium (KV), and calcium channels (CaV). All TRP channel subunits consist of a transmembrane domain (TMD) with six membrane-spanning helices S1-S6, and intracellular amino- and carboxyl- termini [1]. A characteristic of TRP channels is the polymodal gating behavior; these channels are activated by a plethora of physical and chemical stimuli, including membrane voltage, temperature, force, and numerous endogenous and exogenous ligands [1,2]. Thus TRP channels are particularly capable of sensing multiple environmental cues and integrating them into cellular signaling events including calcium signaling [1,2].

Among the six TRPV subfamily members, TRPV1-4 are non-selective, Ca2+-permeable cation channels intrinsically sensitive to warm to hot temperatures (thermoTRPVs) [[7], [8], [9], [10], [11]]. Broadly expressed, these thermally activated channels play important roles in multiple physiological processes, including thermoregulation, osmoregulation and nociception [[12], [13], [14], [15], [16], [17], [18], [19]]. In addition to temperature activation, TRPV1-4 channels are also activated or modulated by a rich array of natural products, synthetic chemicals, and endogenous ligands. For instance, the founding member TRPV1 is activated by extracellular protons, tarantula peptide toxins, and plant products such as capsaicin from hot chili pepper, piperine from black pepper and gingerol from ginger [7,[20], [21], [22], [23], [24]]. 2-Aminoethoxydiphenyl borate (2-APB) activates wild-type TRPV1-3 channels and a TRPV4 variant with mutations of two cytoplasmic residues [25,26]. Cannabinoids from the plant Cannabis sativa and cannabinoid analogs have been identified as agonists for TRPV1-4 channels [[27], [28], [29], [30]]. TRPV4 is activated by plant extract bisandrographolide A from Andrographis paniculata, synthetic compounds GSK1016790A and phorbol ester 4α-PDD, and endogenous lipid metabolites [[31], [32], [33], [34]]. Additionally, mechanical stimuli have been reported to activate TRPV2 and TRPV4 channels [[17], [18], [19],[35], [36], [37]].

To better understand the underlying gating mechanisms by these diverse stimuli and to develop efficient rational therapeutics, we would need high-resolution structures of TRP channels at the atomic level. X-ray crystallography is a well-established technique for atomic structure determination if well-ordered protein crystals are available, but application of crystallography to these integral membrane proteins has proven to be challenging. X-ray crystal structures have been achieved only until very recently for a limited number of TRP channels. In 2013, Cheng and Julius and colleagues published their groundbreaking papers reporting near-atomic resolution structures of TRPV1 determined by single-particle cryo-EM without the need of protein crystals [38,39], which have transformed the field of structural biology of membrane proteins. With the advent of ‘Resolution Revolution’ in cryo-EM [40], near-atomic structures of TRP channels from all subfamilies, including TRPV, TRPA, TRPC, TRPM, TRPML and TRPP, have rapidly emerged. Additionally, distinct conformations representing multiple functional states during channel gating have been determined for a number of TRP channels. The wealth of structural information has enormously advanced our understanding of TRP channel structure and function. This review briefly summarizes exciting progress in structural biology of thermosensitive TRPV1-4 channels and its mechanistic insights into ion permeation and gating mechanisms, and remaining important questions.

Section snippets

Architecture of TRPV channels

TRPV channels share a similar architecture including a characteristic cytoplasmic ankyrin repeat domain (ARD) consisting of six ankyrin repeats and a transmembrane domain (TMD) resembling that of VGICs such as Kv channels [41] (Fig. 1). In the transmembrane region, the peripheral S1-S4 domains, akin to the voltage-sensor domains in VGICs, are attached to the central ion-conduction pore domains in a domain-swapped configuration as seen in many VGICs and other TRP channels (Fig. 1). The

Ion selectivity

Thermosensitive TRPV1-4 channels are non-selective cation channels displaying slightly higher permeability for physiological divalent ions such as Ca2+ than monovalent ions such as Na+ and K+ [7,8,11,49], whereas TRPV5-6 are highly selective for Ca2+ with a permeability ratio of PCa/PNa over 100 [50,51]. To reveal the molecular basis of ion selectivity in these channels, unambiguous determination of ion binding sites along the permeation pathway is necessary. Toward this end, recent X-ray

Gating mechanism

Evident from cryo-EM structures of TRPV1 in distinct functional states, two prominent physical constrictions along the ion permeation pathway, the upper selectivity-filter (SF) gate and the lower helix bundle-crossing (HBC) gate, undergo considerable structural rearrangements during channel opening (Fig. 3a) [38,39]. In the apo state, both the SF and HBC gates are closed. At the SF gate, the side chains of M644 point to the central pore axis and the carbonyl oxygens of G643 form the narrowest

Symmetry reduction

Homotetrameric TRP channels are expected to maintain four-fold symmetric arrangements during gating transitions. Surprisingly, crystal structures of rabbit TRPV2 captured conformations with two-fold symmetry instead [52]. The crystal structure of an engineered rabbit TRPV2 construct in complex with the vanilloid ligand RTX shows more pronounced deviation from four-fold symmetry [52]. RTX binds to all four subunits but the modes of interaction differ in the two adjacent subunits, giving rise to

Channel-lipid interactions

It has been well documented that membrane lipids actively regulate the function of many membrane proteins including TRP channels [[77], [78], [79], [80], [81]], thereby playing a central role in physiology. Single-particle cryo-EM of membrane proteins reconstituted into lipid nanodiscs mimicking biological membranes, has enabled detailed characterization of protein-lipid interactions [82]. Structures of TRPV1 embedded in nanodiscs revealed well-resolved lipid-like densities surrounding the

Conclusions and future perspectives

Recent breakthroughs in single-particle cryo-EM have facilitated exponential growth of the number of near-atomic structures of TRP channels, which have provided unparalleled insights into ion permeation, ligand recognition, lipid regulation, and gating mechanisms. The intriguingly complex conformational landscapes of TRP channels such as reduced symmetry and varying degrees of gate opening have also emerged, as exemplified by TRPV2 and TRPV3 channels. However, fundamental mechanisms such as

Acknowledgements

The author thanks Z. Deng and G. Maksaev for discussion and gratefully acknowledges support from NIH grant R01 NS099341.

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