(Chapter 9 in Hille, and Genome Biology 2011, 12:218 ) Transient Receptor Potential (TRP) Channels: Intrinsic sensors of the cellular environment. (Chapter 9 in Hille, and Genome Biology 2011, 12:218 )

TRP Channel Characteristics Six-transmembrane (6TM) polypeptide subunits. Tetramers form cation-permeable pores. Most cells have multiple TRP channel proteins (ubiquitously expressed). Most subtypes have multiple splice variants.

Large Family of Homologous Proteins (Canonical) (Vanilloid Binding) (Ankarin Repeat) (Melastatin Related) The TRP superfamily contains a growing number of proteins in vertebrates and invertebrates unified by their homology to the product of the Drosophila trp gene, which is involved in light perception in the fly eye (4). On the basis of structural homology, the superfamily can be subdivided into seven main subfamilies: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPP (Polycystin), TRPML (Mucolipin), TRPA (Ankyrin) and TRPN (no mechanoreceptor potential C, NOMPC) (2, 5, 6) (Table 1). All TRP channels comprise six putative transmembrane spans (6 TM) and cytosolic amino (N) and carboxy (C) termini. The length of these cytosolic tails varies greatly between TRP channel subfamilies, as do their different structural and functional domains (Figure 1) (for detailed reviews see References 1, 2). Like other 6 TM pore-forming proteins, functional TRP channels are either homo- or heteromultimers of four TRP subunits (7, 8). It is generally believed that the cation-permeable pore region is formed by a short hydrophobic stretch between TM5 and TM6. (Novel-only in fish) (Polycystin) Owsianik G, et al. Annu Rev Physiol 68:685-717, 2006 (Mucolipin)

Predicted Structural Topology  Predicted structural topology of TRP channels. (a) All channels contain six transmembrane segments (S1 to S6) with a putative pore region (P) between S5 and S6. Amino and carboxyl termini are variable in length and contain different sets of domains. (b) Distribution of domains in selected human TRP channels [103]. The number and composition of domains vary between different TRP channels and are only partially preserved within members of the same subfamily. aa, amino acids; CaM, calmodulin; EF hand, helix-loop-helix Ca2+ binding motif; PH, pleckstrin homology domain; ER, endoplasmic reticulum; NUDIX domain, nucleoside diphosphate linked moiety X-type motif. It is thought that most TRPs function as homotetramers. The formation of heteromultimeric channels between members of the same subfamily or different subfamilies has been described in several cases (such as between the TRPCs), and this could potentially create a wide variety of channels; however, it is debatable whether or not these multimeric channels are formed  Nilius & Owsianik . Genome Biology 12:218, 2011

Membrane topology and functional domains of TRP subunits. Scott Earley, and Joseph E. Brayden Physiol Rev 2015;95:645-690 ©2015 by American Physiological Society

Functions: Sensory transduction Yeast TRP channels perceive and respond to hypertonicity. Nematodes use TRP channels in neuronal dendrites to detect and avoid noxious chemicals. Male mice use pheromone-sensing TRP channels to detect females. TRP channels in humans sense sweet, bitter and amino acid tastes. TRP channels discriminate warmth, heat and cold. Regulate membrane potential

Functions: Sensory transduction + Cation Currents Open in response to depletion of intracellular Ca2+ stores. Activated by stretch of cell membranes. GPCR activation (DAG, IP3). Activated by intracellular Mg2+ and Ca2+

Ionic Conductances as (depending on availability). Bars indicate maximal and minimal values found in literature; TRPM4 and TRPM5 are impermeable to Ca2+ (for TRPM1, values are deduced from Oancea et al. 2009). (B) Fractional Ca2+ current of TRP channels as found in literature (for TRPM3 Oberwinkler personal communication, see Drews et al. 2010). Gees M et al. Cold Spring Harb Perspect Biol 2010;2:a003962

Ca2+-influx mechanisms mediated by TRP channels. Ca2+-influx mechanisms mediated by TRP channels. A: receptor-operated Ca2+ entry (ROCE). Agonist binding to GPCRs stimulates the activity of PLC, which cleaves the membrane phospholipid PIP2 into IP3 and DAG. IP3 binds to IP3Rs on the ER/SR to cause release of Ca2+ into the cytosol. DAG directly activates Ca2+ influx through TRPC3, TRPC6, or TRPC7 channels on the plasma membrane. B: example trace of changes in intracellular Ca2+ following stimulation of a GPCR, showing the phasic increase resulting from ER/SR Ca2+ release and sustained elevation resulting from ROCE. C: store-operated Ca2+ entry (SOCE). Depletion of Ca2+ in the ER/SR causes clustering of STIM1 in the ER/SR membrane proximal to the plasma membrane. STIM1 interacts with Oria1 at the plasma membrane to promote Ca2+ influx. TRPC1, TRPC4, and/or TRPC5 channels, either independently or in association with STIM1/Oria1, may participate in SOCE. D: example of a typical SOCE experiment. Under Ca2+-free conditions, cells are treated with the SERCA inhibitor thapsigargin, causing Ca2+ to leak from the ER/SR into the cytosol. Reintroduction of Ca2+ to the bathing solution after ER/SR stores are depleted causes SOCE. Scott Earley, and Joseph E. Brayden Physiol Rev 2015;95:645-690 ©2015 by American Physiological Society

TRPV4 sparklets in endothelial cells and vascular smooth muscle cells. TRPV4 sparklets in endothelial cells and vascular smooth muscle cells. A: time-lapse image of a typical TRPV4 sparklet recorded from a primary cerebral artery endothelial cell using TIRF microscopy. The cell was stimulated with the selective TRPV4 agonist GSK1016790A (GSK; 100 nM). B: TRPV4 sparklets recorded from a tsA-201 cell transfected with TRPV4 (top) and from a native cerebral artery myocyte (bottom). The middle and right panels demonstrate the single channel-like behavior of these events. TRPV4 sparklets were stimulated with GSK. C: TRPV4 sparklets recorded from the intact endothelium of cerebral arteries from mice expressing the Ca2+-indicator protein GCaMP2 exclusively in the endothelium using high-speed, high-resolution confocal microscopy before and after stimulation with GSK. Experiments were performed in the presence of cyclopiazonic acid (CPA) to eliminate interference from ER Ca2+-release events. Representative traces indicating single channel-like events are shown below. [A from Sullivan and Earley (336). B from Mercado et al. (221). C from Sonkusare et al. (323), with permission from American Association for the Advancement of Science.] Scott Earley, and Joseph E. Brayden Physiol Rev 2015;95:645-690 ©2015 by American Physiological Society

Excitable Cells Express TRPC 1,4, and 6 expressed in pulmonary VSMC. Similar expression in small versus large arteries. Most TRPC subtypes expressed in brain.

TRPC Channel Function Characteristics Unique Isoforms Low selectivity for Ca2+ over Na+ Inhibited by high [Ca2+] Activated by diacylglycerol (DAG) analogues Insensitive to protein kinase C Highly expressed in smooth and cardiac muscle cells Unique Isoforms TRPC1 activated by mGluR1 and contributes to excitatory postsynaptic potential (EPSP) TRPC3 and 1 are putative InsP3 receptor-binding SOC TRPC6 is part of α1-adrenoreceptor-activated cation channel in vein myocytes. TRPC4 knockout mice have defects in lung vasoregulation and microvascular permeability.

TRPC Pore Selectivity: Na+/Ca+ Figure 10   Negatively charged residues located outside of the pore mouth determine permeation properties of TRPC1/5 channels. (A) Putative pore regions of TRPC1 and TRPC5 channels. All negatively charged residues are shown in red, and numbers annotate those residues that are involved in modifications of channel properties. Residues of the pore helix are indicated in green. The region that corresponds to the selectivity filter is in bold italic. (B) Distribution of electrostatic potentials on the surface of putative selectivity filters of TRPC1 (left) and TRPC5 (right). The negative and positive charges are shown in red and blue, respectively. Localization of the main residues is annotated. (C) Whole-cell D633N currents at holding potential −60 mV (left). Averaged I–V values of wild-type TRPC5 and D633N recorded in Na+, Ca2+, and Mg2+-free (NMDG+-containing) extracellular solution (right). (D) A model of the TRPC5-cation conduction pathway at negative (left) and positive (right) potentials. For clarity, only two subunits are shown. D633 and D636 are designated as open circles, whereas Mg2+ is shown as an octagon. The bold arrows indicate the direction of cation fluxes. The thin arrows indicate that Mg2+ is a fast blocker. P, P-helix; S5, TM5; S6, TM6; double plus signs, positively charged; double minus signs, negatively charged. (Panels C and D are adapted from Reference 88, © 2005 by The Society of Neurosciences.)

TRPC Channels: Proposed Mechanisms of Activation Function as receptor-operated channels activated by GPCR, TK and intracellular messengers. Analogous to Drosophila phototransduction TRP. Heteromultimers are distinct from homomultimers. Known multimers TRPC1/TRPC4 TRPC1/TRPC5 TRPC4 monomer TRPC5 monomer DE Clapham; NATURE;426(4) 2003.

TRPV (vanilloid receptor) subfamily General Characteristics Capsaicin (vanilloid compound) is a ligand. Ca2+ permeant channel potentiated by heat and decreased pH Inhibited by intracellular phosphatidylinositol-4,5-bisphosphate (PIP2) TRPV1/2 (50% homology) may mediate high-threshold noxious heat sensation TRPV2 can function as a mechano-sensor in VSMC Required for macrophage early phagocytosis TRPV3 Increased temperature activates (22ºC to 48ºC) as does camphor Neuronal distribution overlaps with TRPV1 suggesting heteromultimers. High expression in skin, tongue and ‘warm sensitive’ neurons. TRPV4 Current potentiated by hypotonicity (cell swelling) and activated by moderate temperature (-/-) mice have impaired renal response to hypertonicity Hypotonicity increases current in primary afferent nociceptive nerve fibers TRPV5/6 Most Ca2+ selective of all TRP channels Constitutively active but Ca2+ inactivation Essential for Ca2+ reabsorption in the kidney

TRPV4 Current Stimulated by Cell Swelling and Heat DAG Heat Arachidonate Hypotonic Saline HEK cells transfected with TRPV4 protein.

TRPM (melastatin) subfamily TRPM1 expression decreased in highly metastatic versus non-metastic melanoma cells Widely expressed but function and electrophysiological properties have not been described. TRPM2 forms a Ca2+-permeant channel fused to an enzymatic ADP-ribose pyrophosphatase gated by ADP ribose and nicotinamide adenine dinucleotide responsive to H2O2 and tumor necrosis factor-a (sensor of intracellular oxidation/reduction) TRPM3 forms a Ca2+-permeant nonselective channel that is constitutively active activity is increased by hypotonicity expressed primarily in kidney TRPM4 and TRPM5 are the only monovalent-selective TRP family ion channels Ca2+-activated nonselective channel Activated by GPCRs coupled to PLC-dependent ER Ca2+ release TRPM5 found in cells expressing taste receptors sweet, umami and bitter taste sensation TRPM6 and TRPM7 contain poorly understood but functional kinase domains Permeant to both Ca2+ and Mg2+ Inhibited by >0.6mM intracellular free Mg2+ TRPM8 nonselective, outwardly rectifying channel activated by cold (8–28 ºC) Activity enhanced by ‘cooling’ compounds such as menthol and icilin. Thought to function as a thermosensor in small-diameter primary sensory neurons

TRPP (polycystin) and TRPML (mucolipin) subfamilies TRPP1, TRPP2 and TRPP3 are the polycystic kidney disease proteins PKD2, PKD2L1 and PKD2L2 These are 6TM Ca2+-permeant channels PKD1 (formerly known as TRPP1 or polycystin-1L1) are 11TM proteins that contain a C-terminal TRP-like domain but aren’t channels. Autosomal dominant polycystic kidney disease is caused by mutations in PKD1 or TRPP1 Mucolipins (MCOLN1, and MCOLN2) are 6TM channels restricted to intracellular vesicles while TRPML3 can localize to the cell membrane. Mutations in TRPML1 are associated with a neurodegenerative lysosomal storage disorder Defects in TRPML3, present in hair cells, causes deafness and pigmentation defects.

Large Family of Homologous Proteins DE Clapham; NATURE;426(4) 2003.

TRP Channels Regulate Ca2+ Entry TRP channels depolarize excitable cells and modulate the driving force for Ca2+ entry. (A) Depolarization of excitable cells upon opening of TRP channels regulates voltage-dependent Ca2+, K+, and Na+ channels. (B) Membrane depolarization by TRP channels results in a reduced Ca2+ entry via ORAI, whereas hyperpolarization of the membrane by BK, IK, or SK channels results in an increased Ca2+ influx. This Ca2+ then modulates TRP and BK, IK, and SK function to fine-tune the [Ca2+]i content. Gees M et al. Cold Spring Harb Perspect Biol 2010;2:a003962

TRP Channels in VSMC Inoue et al. Circ Res. 2006 Jul 21;99(2):119-31

TRP Channels in Neurons TRP channels in growth cone guidance. Growth cones turning in a gradient of an attractive growth factor (i.e., netrin-1; red shading) exhibit more elaborated filopodial structure in which growth factor concentrations are higher. Resting TRP channels (blue rectangles) are activated by receptors for growth factors or other guidance cues (black rectangles) that signal through PLC. Open TRP channels (orange rectangles) permeate Ca2+ directly and indirectly stimulate Ca2+ influx through voltage-dependent Ca2+ channels (Cav; red ovals) by membrane depolarization. Growth factor signaling through PI3-kinase (PI3-K) may also stimulate rapid insertion of TRP channels contained in vesicles (green circles) to augment TRP channel activity. Ca2+ influx presumably establishes a gradient of increased [Ca2+]i (blue shading) across the growth cone that is instructive for turning. Ramsey, et al. Ann Rev Physiol 68:619-647, 2006

TRP Channels in Organelles Expression of TRP channels in intracellular compartments. Early endosomes (EE) are derived from the plasma membrane via endocytotic vesicles (EV). The cargo from these early endosomes can either go back to the plasma membrane via the recycling endosomes (RE) or follow the late endocytotic pathway via late endosomes (LE) to the lysosomes (LY). Intermediate transport vesicles (TV) are derived from the ER and/or the trans-Golgi-network (TGN). The content in these transport vesicles can either be delivered to early endosomes, late endosomes, or be transported to the plasma membrane via secretory vesicles (SV) or secretory granules (SG). Synaptic vesicles (SyV) are derived from early endosomes and release neurotransmitters in the extracellular space. Only intracellular locations of TRP channels are indicated. Gees M et al. Cold Spring Harb Perspect Biol 2010;2:a003962

Summary TRP channels are important for Ca2+ entry TRP channels regulate voltage-dependent channels TRP channels regulate the driving forces for Ca2+ via other channels TRP channels are targets of Ca2+ TRPs are intracellular channels and may also act as scaffolding proteins

A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades. C Wilson and SE Dryer. Am J Physiol Renal Physiol 306: F1018–F1025, 2014 Questions: Is TRPC6 mechanosensitive in all cell types examined? Do all perturbations that affect DAG activation similarly affect stretch activation? What was the rationale for using CHO-K1 cells in this study? Do you think this is a valid justification (why or why not)? Why were hypoosmotic solutions used to evaluate mechanosensitivity of the currents? Would a hyperosmotic solution have the same effect? Do you agree with the authors that the traces in Fig. 1 show that the currents elicited by the hypoosmotic solution are similar to those elicited by OAG or ATP? What is the conclusion(s) from the data in Figures 2 and 3? What do GDP- βS, SKF96365 and LA3+ test for? Would you expect OAG to be affected by any of these agents?

Do TRPC-like currents and G protein-coupled receptors interact to facilitate myogenic tone development? How are the channels transfected into the CHO cells in Figure 5 different from those transfected in Figures 1 – 4? What do you know about the residue N143? Fig. 5 shows relative expression of the two isoforms and channel activity. Do these studies specifically determine the effect of the N143S mutation on TRPC6 activation? What is the conclusion drawn from Fig. 5 data? Figure 6 is testing for dominant negative effects of the mutation. If the channel is dominant negative, what would be seen? Is this what is observed? What are the overall conclusions from the study? Do they offer any insights into how these channels are activated or inactivated?

Summary of Article We conclude that mechanical activation of TRPC6 channels proceeds by biophysical mechanisms that are distinct from those used in G protein signaling cascades, and these observations strongly suggest that wild-type TRPC6 channels are intrinsically mechanosensitive.