The Molecular Diversity of TRP Channels and Related Proteins

Introduction

TRP channels are a large family (20 genes) of plasma membrane, non-selective cationic channels that are either specifically or ubiquitously expressed in excitable and non excitable cells. These proteins are divided into three main subfamilies on the basis of sequence homology; TRPC, TRPV and TRPM1 (see Table). Like the K_V channel family, the TRP family can also form heteromers consisting of different members of the same subfamily. Their non-selective cationic nature makes them depolarizing agents, while their calcium permeability makes them as transducers leading to [Ca²⁺]_in elevation. However, there are many different gating mechanism and/or modulating agents that activate and inactivate different members of this channel family (for reviews see^2,3).

TRP channels belong to the superfamily of cation channels with six transmembrane segments (see Figure 1). This superfamily includes voltage-gated K⁺, Na⁺, and Ca²⁺ channels, as well as cyclic nucleotide gated (CNG) channels, hyperpolarization activated cyclic nucleotide gated (HCN) channels, and the most closely related group, polycystins (PKD)^4,5. TRP channels contain specific ankyrin domains in the cytosolic N-terminal portion and proline-rich motif in the cytosolic C-terminal portion.

Gating mechanism of TRP channels

The TRPC subfamily channels are all activated by products of the Receptor-Gq-PLC signal transduction pathway (see below, for review see²). However, the link between receptor activation and channel gating is very controversial. This point can be demonstrated in TRPC3 channel activation. One report shows that TRPC3 interacts directly with an ER membrane channel, IP₃R, thus displacing calmodulin that inhibits the channel and therefore activating TRPC3⁶. Another report, using IP₃R knockout cells, shows that muscarinic receptor activation via PLC and DAG, independently of IP₃R, activates TRPC3⁷. In addition, many TRPC channels are believed to be activated as a result of depletion of calcium from the ER/SR (I_CRAC).

Thapsigargin (#T-650), an inhibitor of the ER/SR ATPase is often used to induce store depletion, and to examine TRP channel activation, in response to this treatment.

Three other members (from the other two subfamilies: TRPV1⁸, TRPV2⁹ and TRPM8^10,11) are gated directly by changes in temperature (with ranging sensitivities). With their specific expression in sensory neurons these are at the moment strong candidates for the physical transducers of cold and heat into a form of cellular signal (either voltage or calcium or both). Another member, TRPV4, forms mechano/osmolarity sensitive channels and therefore may function as a transducer serving both in cell volume and sensory sensation regulation. TRPV5 and TRPV6, which are both found in epithelial cells are activated by reduction in the level of the intracellular calcium concentration and therefore may bear a similar gating mechanism to the TRPC family (i.e., store depletion).

Channels from the TRPM subfamily were reported to be gated by ADP-ribose (see² TRPM2) and phosphorylation (TRPM7, a protein which consists of a kinase attached to a channel)¹² and by low temperature (TRPM8, see above). The gating of other members of the TRPM subfamily is still unknown.

Classification of TRP channels

The search of Caenorhbditis elegans genome database revealed 13 TRP genes that can be divided into three families¹³. The first is a family of short TRP (TRPC), genes which have a reading frame of ~900 residues. The more distant family, TRPV (OTRPC, originally named after the homology with osm-9, the first cloned member of this family) also has a ~900 residue reading frame. The third family, TRPM (long TRP), has a reading frame of ~1600 residues.

The suggested classification, based on C. elegans genome, seems to apply widely¹³. The Drosophila TRP and TRPL channels thus belong to the TRPC family.

To date, one toxin, Maitotoxin, extracted from a marine dinoflagellate was described to be a specific activator of human TRPC1¹⁴.

Physiological role of TRP channels

The cytosolic Ca²⁺ serves as an intracellular mediator for many extracellular signals. The receptor coupling via heterotrimeric G proteins to different isoforms of phospholipase C (PLC) leads to the breakdown of phosphoinositide to inositol 1,4,5-phosphate (InsP₃) and diacylglycerol (DAG). After this reaction, a biphasic increase in cytosolic Ca²⁺ concentration occurs. The first stage results from transient InsP3-mediated release from intracellular stores. The second, more sustained phase, results from store-operated activation of Ca²⁺ -permeable membrane channels. The suggested role of this phase is in refilling the intracellular Ca²⁺ stores or prolonging the response^15-17.

Depending on the cell type, the second-phase Ca²⁺ channels may vary from highly selective Ca²⁺ channels to nonselective Ca²⁺-permeable channels¹⁸. The best studied store-operated Ca²⁺ current is Ca²⁺-release activated Ca²⁺ channel (CRAC), originally described in mast cells and T lymphocytes^19,20.

The first step for molecular identification of store-operated Ca²⁺ channels came from the study of Drosophila photoreceptors. In insects, in contrast to vertebrates, light activation of rhodopsin is coupled via a G protein to PLC, which leads to a biphasic Ca²⁺ entry. A mutant called trp (transient receptor potential) was shown to lack the second phase, light induced current (LIC). It has been shown that trp codes for a Ca²⁺-selective channel protein, TRP. Later, another closely related channel, TRPL, was cloned. It forms a less-selective Ca²⁺-permeable channel^21,22.

Most data for mammalian TRP channels is based on heterologous expression system. It is generally agreed that TRP channels are activated downstream of G-protein-coupled receptors, which induce PLC-mediated phosphoinositide breakdown. However, the downstream signaling pathways that finally activate TRP channels remain highly controversial. For nearly all of the functionally expressed TRP channels, there is at least one report proposing a store-operated mechanism of activation^23-29. On the other hand, there is growing evidence for the involvement of store in dependent pathways in the regulation of TRPC3^30-33, TRPC5³⁴, TRPC6^{33, 35} and TRPC7³⁶. For TRPL, TRPC3 and TRPC6, direct activators have been identified that stimulate the channels in a membrane-confined manner. Polyunsaturated fatty acids were shown to gate Drosophila TRPL³⁷ and DG to activate TRPC3, TRPC6, and TRPC7^{33, 36}. Recently, by using knockout mice, TRPC4 was shown to correspond to the store operated calcium channel in endothelial cells, which controls vasorelaxation in blood vessels³⁸.

The C. elegans member of the TRPV (OTRPC) family, osm-9 (homologue of TRPV4), is involved in responses to odorants, high osmotic strength, and mechanical stimulation³⁹. Similarly, the mammalian members of this family, TRPV1 (VR1)⁸ and TRPV2 (VRL1)⁹, form non-selective Ca²⁺-permeable channels activated by heat and other pain-producing stimuli⁴⁰. Other members of this family, TRPV6 (CaT1, ECaC)^41,42 and TRPV5 (CaT2)⁴³, seem to be involved in Ca²⁺ transport in epithelial cells⁴⁴.

The function of the TRPM family channels is largely unknown. One mammalian TRPM channel, TRPM1 (melastatin), is found in melanocytes and its level is decreased in metastatic cells⁴⁵. Another one, called TRPC7, which is different from the TRPC7 belonging to TRPC family³⁷, has been recently described⁴⁶. Another one, named MTR1, has been recently cloned⁴⁷. Many TRPM channels are reported to be involved in cell growth and differentiation.

Localization of TRP channels

The tissue localization of TRPC differs greatly among the family members (see Table). TRPC1 is expressed ubiquitously, while TRPC3, TRPC4 and TRPC5 are almost exclusively localized in the brain²⁹. In rat, TRPC2 is exclusively expressed in the vomeronasal organ⁴⁸. In humans TRPC2 is a pseudogene, possibly in agreement with the lack of vomeronasal organ in higher primates⁴⁹. Interestingly, mice lacking the TRPC2 gene failed to express male-male aggression and did not distinguish between male and female in their sexual behaviour⁵⁰.

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