Overview: TRP Channels: Types, Distribution, and Function

Transient receptor potential (TRP) channels are a large family of cation-specific ion channels that have acquired immense attention, due to their involvement with a variety of cellular functions. TRP multigene family sequence is conserved, both in vertebrates and invertebrates [1,2] and member proteins are classified in subfamilies named TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), TRPP (polycystin), and TRPN (NOMPC-like; Figure 15.1). Among the subfamilies, 28 members are expressed in vertebrates (with the exception of TRPN) of which 27 are found in humans (excluding TRPC2, which is encoded by a pseudogene) [1,2] (Figure 15.1). TRP channels are expressed in a range of different human cell types and are tied to a variety of cellular processes ranging from stimuli sensation to ion homeostasis [3].

In this chapter, we plan to provide an overview of TRP channels in humans, emphasizing on the function and dysfunction of the cerebral circulatory system. The TRPC subfamily consists of six members (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7), which are robustly expressed in the human brain. In regard to the vasculature, TRPC1 displays a ubiquitous distribution pattern, whereas C3, C4, and C6 are expressed in vascular smooth muscle cells and C4 in the endothelium (Figure 15.1). These channels are engaged in brain development, cell differentiation, mechanosensation, arterial tone control, smooth muscle cell proliferation, and angiogenesis (for detailed review, see Ref. [2]). The human TRPV subfamily also contains six members (TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6), all of them being represented in the brain tissue (Figure 15.1). V2 subunit was found in smooth muscle cells and V3, V4 in the vascular endothelium, playing key roles in thermosensation, mechano/osmolarity sensation, calcium reabsorption, and synaptic adaptation [2,3]. In humans, there are eight members of the TRPM subfamily (TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, and TRPM8) among which M7 is distributed ubiquitously, whereas M2, M3, and M5 variants are expressed in the brain and M4 and M8 are also found in the smooth muscle cell layer [2] (Figure 15.1). These subunits are thought to play an integral part in myogenic tone development, apoptosis, cell cycle control, thermosensation, and Mg^2 + homeostasis [2,3]. The TRPA subfamily is comprised of a sole member (TRPA1) (Figure 15.1), typically associated with thermosensation, olfactory responses, and endothelium-dependent dilation [2,3]. The TRPML subunit consists of TRPML1, TRPML2, and TRPML3 (Figure 15.1); however, there is no evidence of TRPML expression in the resistance vasculature of the brain [2,3]. On the other hand all three members of the TRPP subfamily (TRPP2, TRPP3, and TRPP5) are present in arteries. These subunits have been linked to pressure and flow sensing along with the maintenance of vessel wall integrity [2,3].

TRP Channels in Diseases

TRP channels are associated with a variety of physiological processes in humans including signal transmission, nociception, thermosensation, osmolarity regulation, and arterial tone development [4]. TRP channel activity is triggered by changes in cellular temperature, pressure, chemical agents, osmolarity, and pH [4,5] and by naturally occurring spices, herbs, toxins, and venoms [4,6]. Changes in intercellular calcium (Ca^2 +), induced by the activation of G-protein coupled receptors, phosphatidylinositol 4,5-bisphosphate, diacylglycerol (DAG), adenosine triphosphate (ATP), and calmodulin are also known to influence TRP channel activities [4,7]. In addition, interactions of TRP channels with intercellular proteins forming “channelosomes” are now believed to effect its trafficking, positioning, and activity [4,8,9]. Consequently, mutations in TRP channel encoding genes may be responsible for several diseases of the cerebrovascular and cardiovascular systems [4]. TRPC3, TRPC5, TRPC6, TRPM2, TRPM4, TRPM7, and TRPML1 have been implicated in CNS disorders, whereas TRPC1, TRPC3, TRPC6, and TRPM4 have been linked to cardiovascular abnormalities [4]. Although human genetic studies have demonstrated the role of TRP gene mutations in at least 12 hereditary diseases (e.g., polycystic kidney disease, hereditary motor and sensory neuropathy, spinal muscular atrophy, and progressive familial heart block), transgenic and knockout animal experiments, on the other hand, have revealed the role of TRP channels in the development of disease pathologies [4,10]. By targeting TRP channels, novel treatment strategies could be deducted for these so-called TRP channelopathies, a rapidly expanding category of diseases. TRP channel targeting molecules have been developed and tested clinically in recent years with minimum success [11]. For example, TRPV1 antagonist underwent clinical trials for the treatment of inflammatory, visceral, and neuropathic pain; however, this resulted in adverse side effects such as hyperthermia and impaired heat sensation in patients [11].

TRP Channels in Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) account for the majority of blood vessel wall composition and hence are widely distributed, playing major roles in circulatory, respiratory, gastrointestinal, and urogenital systems [12,13]. Although blood flow regulation by contracting and relaxing the blood vessel remains the primary function of VSMCs, they have been implicated in many other physiological functions such as proliferation and migration, production of cytokines/chemokines/matrix proteins/growth factors/cell surface adhesion molecules, and in antigen presentation [12–17]. Smooth muscle activity, in large part, is determined by the changes in Ca^2 + concentration, a process dependent on Ca^2 + entry from the extracellular space through ion channels and on the Ca^2 + release from the intracellular calcium stores [12]. Extracellular Ca^2 + entry is facilitated by voltage-operated calcium channels (L-type and T-type) and voltage-independent calcium channels (receptor-operated channels, store-operated channels (SOC), and stretch-activated channels). TRP channels have been reported to be a key component of the voltage-independent calcium channels in the SMCs; their dysfunction is responsible for vascular pathologies ranging from arterial hypertension and arthrosclerosis, to stroke and cardiovascular diseases [12,18].

TRP Channels and Hypertension

Hypertension is considered a primary risk factor of vascular diseases and has been implicated in atherosclerosis, stroke, heart failure, and renal complications [64,65]. Among the several factors that contribute to the development of hypertension, dysregulation of Ca^2 + homeostasis and the consequent vascular dysfunction are most common. Advances in the TRP channel field have enabled us to understand that these cation channels, and variations in their expression and/or regulation, are responsible for the development of many disorders, including hypertension. The TRP channel subunits TRPC1, TRPC3, TRPC5, TRPC6, TRPV1, TRPV4, TRPV5, TRPM4, TRPM6, TRPM7, and TRPA1 have all been connected to the development and/or progression of hypertension. TRPC3, TRPC5, and TRPM6 were found to be reduced significantly in patients after hypertensive intracerebral hemorrhage in comparison with controls [66]. In addition, the authors also found a correlation between the expression of TRPC3 and hypoxia inducible factor 1a, indicating its association with hypoxic conditions in the human cerebral tissue [66]. In spontaneously hypertensive rats, the expression of TRPC1, C3, and C5 were significantly increased in the mesenteric arteries and was associated with an increase in norepinephrine-induced vasomotion in comparison with normotensive rats [67]. Chronic treatment with candesartan or telmisartan significantly reduced the expression of these TRPs and norepinephrine-induced vasomotion in these animals [67]. Activation of the TRPC3 channel leads to myocyte depolarization and Ca^2 + entry through VDCC resulting in vasoconstriction [68]. VDCC Ca^2 + entry is suggested to be crucial for the autoregulation of cerebral blood flow and hence could be an interesting target to address dysfunctional vascular contractility, as during hypertension [68]. In addition, Xi et al. have demonstrated a mechanism of IP3-induced vasoconstriction of cerebral arteries, which occurs independent of SR Ca^2 + release and mediated by TRPC3 channels and IP3 receptor-dependent I(Cat) activation [69]. Monocytes of erythropoietin-treated, hemodialysis patients revealed an increased expression of TRPC5 mRNA accompanied by increased systolic blood pressure, indicating a role of TRPC5 in erythropoietin induced hypertension [70]. In HUVECs, TRPC6 and TRPV1 mRNA expression levels were significantly increased after exposure to a pulsatile atheroprone shear stress flow in comparison to an atheroprotective flow profile [71]. Furthermore, the authors also found an association between TRPC6 mRNA expression and tumor necrosis factor-α mRNA in the human vascular tissue, delineating its connection to the inflammatory processes in the vasculature [71]. Recently, the involvement of TRPC6 and 20-hydroxyeicosatetraenoic acid (HETE) in the autoregulation of cerebral blood flow was elucidated under conditions of hypertension, where the authors described the loss of autoregulation in aged, Ang II-induced hypertensive mice along with exacerbated disruption of the BBB, increased neuroinflammation, and impaired hippocampal-dependent cognitive function [72].

In addition, it has been shown that 20-HETE activation of TRPV1, in part, mediates pressure-induced myogenic constriction and underlines 20-HETE-induced elevations in blood pressure and coronary resistance [73]. The connection between TRPV1 and intraluminal pressure changes were first described by Scotland et al. as an elevation in intraluminal pressure generated 20-HETE, activating TRPV1 on C fiber nerve endings and resulting in a myogenic response [74]. TRPV1 activation by dietary capsaicin enhanced endothelium dependent relaxation in mice, an effect absent in TRPV1 knockout mice [75]. Furthermore, chronic stimulation of TRPV1-activated PKA, resulted in increased eNOS phosphorylation, improved vasorelaxation, and the lowering of blood pressure in genetically hypertensive rats [75]. Remarkably, long-term administration of capsaicin significantly delayed the onset of stroke and increased survival time in spontaneously hypertensive rats, an effect mediated through eNOS-dependent vasorelaxation and induced by TRPV1 activation [76]. Moreover, long-term activation of capsaicin increased ATP binding cassette transporter A1 and reduced low-density lipoprotein-related protein1 expression in the aorta of atherosclerotic mice (ApoE^−/−), reducing lipid storage and atherosclerotic lesions in the aorta, an effect absent in ApoE(^−/−)/TRPV1 (^−/−) double knockout mice [77].

Rodent experiments have suggested activation of the sympathetic nervous system through the osmosensitive channel TRPV4 to be responsible for the robust increase in blood pressure after water ingestion, observed in human subjects with impaired baroreflex function [78]. Even though TRPV4 has been shown to be involved in pulmonary vasoreactivity [79] and hypoxia-induced pulmonary hypertension [80], and that nitric oxide synthase-inhibition induced hypertension is exacerbated in TRPV4 knockout mice [81], its role in the cerebral vasculature and its dysfunction, remains to be clearly elucidated. The inhibition of WKN4, a protein serine/threonine kinase that positively regulates the TRPV5-mediated Ca^2 + transport process, is believed to be involved in the pathogenesis of familial hyperkalemic hypertension [82]. Moreover, TRPV5 knockout mice present phenotypic defects of renal disease such as hypercalciuria and impaired bone mineral density, exemplifying the role of this channel in renal physiology, hypertension, and genetic disorders of the kidney [82,83].

The role of TRPM subunits in hypertension is mainly represented by TRPM4, 6, and 7, where TRPM4 was first shown to be increasingly expressed in ventricular samples of spontaneously hypertensive rats in comparison with normotensive controls [84]. Interestingly, TRPM4 has been also connected with the electrophysiological perturbations associated with cardiac hypertrophy, of which hypertension is an important risk factor [85]. TRPM4 knockout mice present dysregulation of blood pressure toward hypertensive levels as TRPM4 regulates catecholamine release from chromaffin cells, the dysfunction of which leads to increased sympathetic tone and hypertension [86].

Mg^2 + influences many cellular functions and variations in Mg^2 + in the serum and tissue has been known to inversely affect blood pressure [87]. As TRPM6 and TRPM7 are associated with Mg^2 + transport in the tissue, they are also involved in the development of vascular dysfunctions and hypertension. Whereas TRPM6 is expressed in epithelial cells, TRPM7 is globally expressed and is known as an important regulator of Mg^2 +. Membrane-associated Ang II- and aldosterone-regulated TRPM7 are involved in the DNA and protein synthesis in both rodent and human VSMCs [88]. Importantly, the expression of TRPM7 was blunted in spontaneously hypertensive rats, which resulted in decreased Mg^2 + in the VSMC [89]. Similarly, in low intracellular Mg^2 +-exhibiting mice, the expression of TRPM7 was significantly elevated in the vasculature, whereas its downstream target, anti-inflammatory annexin-1, was reduced [90]. These animals also exhibited endothelial dysfunction, changes in vascular structure, and inflammation, revealing the role of TRPM7 signaling in the maintenance of vascular integrity and function [90]. A recent investigation has also revealed the role of TRPM7 in vascular remodeling, where its expression increased with the pressure overload-induced vascular wall thickening [91]. It was also associated with simultaneous accumulation of macrophages in the adventitia and decreased expression of annexin-1, an effect reversed by TRPM7 inhibitor 2-aminoethoxydiphenyl borate [91].

TRPA1 are a class of mechanosensitive Ca^2 + permeable channels that are activated by irritant chemicals and pungent natural compounds [92,93]. L-type calcium channel antagonist 1,4-dihydropyridines, including nifedipine, nimodipine, nicardipine, and nitrendipin, commonly used in the treatment of hypertension, have been shown to exert powerful excitatory effects on TRPA1 channels [92]. In addition, nifedipine is known to induce large elevations of Ca^2 + in mouse nociceptors expressing TRPA1, an effect abrogated in the TRPA1 knockout mice [92].

TRP Channels and Stroke

Ischemia refers to the inadequate supply of oxygen and nutrients to an organ, resulting in cell death and degeneration. Stroke is the fourth major cause of death and is the leading cause of adult disability in the Western population [94]. As stroke results from the blockage or disruption of a supplying blood vessel, vascular dysfunction is a vital risk factor in its development and during recovery. Hypoxia resulting from ischemia, activates a myriad of cellular responses including the rise in intercellular Ca^2 + [95]. As the rise in intercellular Ca^2 + depends on the activation of Ca^2 + permeable channels and/or by the activation of intercellular Ca^2 + stores, TRP channels, with their known influence on these mechanisms, are likely to play a key role. The first demonstration of the involvement of a TRP subunit in hypoxia was the observation that TRPM7 suppression blocked Ca^2 + uptake, reactive oxygen species production and anoxic cell death in cortical neurons subjected to prolonged oxygen glucose deprivation [96]. Electrophysiological investigation on acute hippocampal slices showed that TRP channels are activated by cellular stress, contributing to ischemia-induced membrane depolarization, intracellular calcium accumulation, and cell swelling [97]. Likewise, TRPM2 mRNA expression was increased at 1 and 4 weeks following ischemic injury in a rat middle cerebral artery occlusion model of stroke [98]. In mouse hippocampal neurons, it was demonstrated that TRPM7 channels were involved in detecting the reduction in extracellular divalent ions, known to contribute to neuronal death after brain ischemia [99]. TRPM7 suppression by shRNA made neurons resistant to neuronal damage after brain ischemia, preserving neuronal morphology and function, leading to long-term potentiation and animal survival [100]. However, an assessment of 16 tag-single-nucleotide polymorphisms of the TRPM7 gene in humans showed no evidence for its role as a risk factor for ischemic stroke incidence [101].

An increase in TRPV4 expression in the cortical and hippocampal astrocytes of adult rat was shown to coincide with the development of astrogliosis after ischemia [102]. In addition, cultured hippocampal astrocytes have been found to respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate as detected by an increase in intracellular Ca^2 +; this effect was augmented after hypoxic/ischemic injury, demonstrating the possible role of TRPM4 in astroglial reactivity after ischemia [102]. An investigation on a primate model of stroke has revealed promising results that Tat-NR2B9c, a PSD-95 interfering peptide known to disrupt TRPM7/PSD-95/neuronal NOS pathway, reduced stroke volume, maintained gene transcription, and preserved neurological function after ischemia [103,104]. The emerging role of TRP channels in neuronal injury development after stroke opens up a potential paradigm for clinical interventions in patients, extending even beyond the acute phase of stroke. However, the expressional, regulatory and functional characteristics of the many different TRP subunits need to be thoroughly elucidated so that a feasible therapeutic strategy could be derived for interventions in stroke pathology.

Future Directions

Our understanding of TRP channels has exploded in the past decade (> 1600 manuscripts annually [105]), fueled by its potential as a drug target. However, the conundrum surrounding TRP channel functionality and its connection to human disease is currently underdeveloped. The role of TRP channels in the cerebral circulation and in cerebrovascular diseases is still in its infancy. As TRP channels have been implicated in myogenic tone blood flow control along with the permeability of the BBB, collateral formation, angiogenesis and vascular remodeling, its significance in the cerebral circulation and cerebrovascular disorders have gained appreciation. The varied and global expression of TRP channels demonstrate its multifaceted roles in cellular physiology and at the same time presents itself as a challenging barrier for drug design and implementation. This task is further complicated by the ability of TRPs to heteromultimerize and to interact with other intercellular proteins (channelosomes). The fact that many of TRP members are activated by natural compounds (e.g., carvacrol, capsaicin) is enthralling, as plant extracts have been used in traditional medicine for many centuries. Hence, it is conceivable that the regulation of TRP channels and subsequent intervention of their associated pathologies may be possible through dietary supplements via naturally occurring compounds, thereby minimizing the risk of side effects in patients. Given the ubiquitous nature and the presence of multidimensional activation pathways, a comprehensive understanding of TRP channel biology remains the “sine qua non” for extracting its therapeutic potential.