Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.     

Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties

Heat-sensitive transient receptor potential (TRP) channels (TRPV1-4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein-tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a "spectra FRET" approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity.     

TRP channels in disease

"Transient receptor potential" cation channels (TRP channels) play a unique role as cell sensors, are involved in a plethora of Ca(2+)-mediated cell functions, and play a role as "gate-keepers" in many homeostatic processes such as Ca(2+) and Mg(2+) reabsorption. The variety of functions to which TRP channels contribute and the polymodal character of their activation predict that failures in correct channel gating or permeation will likely contribute to complex pathophysiological mechanisms. Dysfunctions of TRPs cause human diseases but are also involved in a complex manner to contribute and determine the progress of several diseases. Contributions to this special issue discuss channelopathias for which mutations in TRP channels that induce "loss-" or "gain-of-function" of the channel and can be considered "disease-causing" have been identified. The role of TRPs will be further elucidated in complex diseases of the intestinal, renal, urogenital, respiratory, and cardiovascular systems. Finally, the role of TRPs will be discussed in neuronal diseases and neurodegenerative disorders.     

TRP proteins and cancer

Cancer is the second most common cause of death in western countries. It is therefore of fundamental importance to improve the treatment of patients with malignant tumors. This goal can only be achieved if we get closer insight in the various mechanisms leading to tumor formation. Significant progress in the understanding of carcinogenesis has been made during the last couple of years. Ion channels contribute to the regulation of cell proliferation which has initially been shown for K+ channels. Meanwhile, other ion channels such as Cl-, Na+ and Ca2+ channels seem to influence cellular function like growth, migration and invasion. In addition, cation channels of the transient receptor potential (TRP) superfamily are implicated in cancer formation. Most recent data concerning TRP vanilloid (TRPV) type 6, TRP melastatin (TRPM) type 1 and 8 channels and their relevance for common human cancer types will be highlighted in this review. Furthermore, TRP channel structure and function will be discussed in the light of their possible importance as prognostic markers and targets for drug discovery.     

Single amino acid change in the fifth transmembrane segment of the TRP Ca2+ channel causes massive degeneration of photoreceptors

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.     

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.     

Transient receptor potential (TRP) channel function in the reproductive axis

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic–pituitary–gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca²⁺ concentration via its own Ca²⁺ permeability and via the activation of voltage-gated Ca²⁺ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.     

TRP channels in disease

“Transient receptor potential” cation channels (TRP channels) play a unique role as cell sensors, are involved in a plethora of Ca²⁺-mediated cell functions, and play a role as “gate-keepers” in many homeostatic processes such as Ca²⁺ and Mg²⁺ reabsorption. The variety of functions to which TRP channels contribute and the polymodal character of their activation predict that failures in correct channel gating or permeation will likely contribute to complex pathophysiological mechanisms. Dysfunctions of TRPs cause human diseases but are also involved in a complex manner to contribute and determine the progress of several diseases. Contributions to this special issue discuss channelopathias for which mutations in TRP channels that induce “loss-“ or “gain-of-function” of the channel and can be considered “disease-causing” have been identified. The role of TRPs will be further elucidated in complex diseases of the intestinal, renal, urogenital, respiratory, and cardiovascular systems. Finally, the role of TRPs will be discussed in neuronal diseases and neurodegenerative disorders.     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Formation of novel TRPC channels by complex subunit interactions in embryonic brain

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.     

Splice Variants of Na(V)1.7 Sodium Channels Have Distinct β Subunit-Dependent Biophysical Properties

Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na_V1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na_V1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na_V1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na_V channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.     

Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK(Ca)) channels are tetramers of alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK(Ca) channels affinity-purified from rat brain to analyze in vivo BKalpha phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKalpha differentially modulates the voltage- and Ca(2+)-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK(Ca) channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKalpha structure and function.     

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca²⁺ and Mg²⁺ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.     

Structural biology of thermoTRPV channels

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.     

Structural Pharmacology of TRP Channels

Transient receptor potential (TRP) ion channels are a super-family of ion channels that mediate transmembrane cation flux with polymodal activation, ranging from chemical to physical stimuli. Furthermore, due to their ubiquitous expression and role in human diseases, they serve as potential pharmacological targets. Advances in cryo-EM TRP channel structural biology has revealed general, as well as diverse, architectural elements and regulatory sites among TRP channel subfamilies. Here, we review the endogenous and pharmacological ligand-binding sites of TRP channels and their regulatory mechanisms.     

A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory Transduction-Associated TRP Channels in Mammals

Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes.     

TRPC1 and TRPC5 form a novel cation channel in mammalian brain

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.     

TRP channels as candidates for hearing and balance abnormalities in vertebrates

In this review, we summarize the potential functional roles of transient receptor potential (TRP) channels in the vertebrate inner ear. The history of TRP channels in hearing and balance is characterized at great length by the hunt for the elusive transduction channel of sensory hair cells. Such pursuit has not resulted in unequivocal identification of the transduction channel, but nevertheless revealed a number of candidates, such as TRPV4, TRPN1, TRPA1, and TRPML3. Much of the circumstantial evidence indicates that these TRP channels potentially play significant roles in inner ear physiology. Based on mutations in the corresponding mouse genes, TRPV4 and TRPML3 are possible candidates for human hearing, and potentially also balance disorders. We further discuss the role of the invertebrate TRP channels Nanchung, Inactive, and TRPN1 and how the functional analysis of these channels provides a link to vertebrate hearing and balance. In summary, only a few TRP channels have been analyzed thus far for a prospective role in the inner ear, and this makes the search for additional TRPs associated with inner ear function quite a tantalizing endeavor.