Ca²⁺ Regulation of TRP Ion Channels

Abstract

Ca²⁺ signaling influences nearly every aspect of cellular life. Transient receptor potential (TRP) ion channels have emerged as cellular sensors for thermal, chemical and mechanical stimuli and are major contributors to Ca²⁺ signaling, playing an important role in diverse physiological and pathological processes. Notably, TRP ion channels are also one of the major downstream targets of Ca²⁺ signaling initiated either from TRP channels themselves or from various other sources, such as G-protein coupled receptors, giving rise to feedback regulation. TRP channels therefore function like integrators of Ca²⁺ signaling. A growing body of research has demonstrated different modes of Ca²⁺-dependent regulation of TRP ion channels and the underlying mechanisms. However, the precise actions of Ca²⁺ in the modulation of TRP ion channels remain elusive. Advances in Ca²⁺ regulation of TRP channels are critical to our understanding of the diversified functions of TRP channels and complex Ca²⁺ signaling.

1. Introduction

Transient receptor potential (TRP) ion channels are a large family of cation channels consisting of six mammalian subfamilies: TRPV, TRPM, TRPA, TRPC, TRPP and TRPML [1]. They are activated by a vast array of stimuli in the environment, ranging from temperature, natural chemicals and toxins, to mechanical stimuli. They also respond to endogenous agents and messengers produced during tissue injury and inflammation [2]. TRP channels therefore function as molecular sensors responsible for detecting the external world and internal milieu, and have been implicated in diverse physiological processes and diseases, such as temperature sensation, vision, taste, pain, itch and cardiovascular diseases [1,2,3,4].

The TRPV subfamily consists of six members. Of them, TRPV1–4 are mainly expressed in the sensory ganglia and skin responsible for thermo-sensation, pain and itch, whilst TRPV5 and TRPV6 are primarily expressed in the kidney and gastrointestinal tract and play a major role in Ca²⁺ absorption and homeostasis [3,5,6]. The TRPM subfamily is the largest, consisting of eight members broadly expressed in a variety of cells and tissues, such as sensory ganglia, pancreatic beta cells, immune cells, the tongue, heart and kidney, and are critical for sensory physiology (e.g., heat (TRPM3), cold (TRPM8), taste (TRPM5) and light (TRPM1) sensing and detection) [5], insulin release (TRPM2, TRPM4 and TRPM5) [7,8], Mg²⁺ homeostasis (TRPM6 and TRPM7), ischemic injury and inflammatory responses (TRPM2, TRPM4) [9]. TRPA1 is the sole member of the TRPA subfamily, implicated in pain, itch and inflammation [10]. TRPC is another big TRP subfamily that includes seven subtypes widely expressed in the central nervous system and various other tissues participating in diverse conditions such as neuron development and epilepsy [5,11]. TRPP channels mainly cause polycystic kidney disease due to mutations in the channel genes. In contrast to other TRP subfamilies largely expressed on cell membranes, TRPML channels are mainly localized in endosomes and lysosomes critical for lysosome storage disorder, vesicle trafficking and ion homeostasis [12]. These TRP ion channels are modulated by various mechanisms, most commonly by membrane voltage, phosphoinositides, phosphorylation and G-protein coupled receptor signaling [5].

Notably, most TRP ion channels are permeable to Ca²⁺ except TRPM4 and TRPM5 [2]. Activation of TRP channels therefore also serves as an important Ca²⁺ entry pathway contributing to fluctuations in intracellular Ca²⁺ ([Ca²⁺]_i) and subsequent Ca²⁺ signaling. Ca²⁺ is a ubiquitous second messenger participating in numerous signal transduction pathways involved in biological processes as diverse as secretion, gene transcription and cell death [13]. Furthermore, similar to other cations, Ca²⁺ ions contribute to the electrochemical gradient in excitable cells and are critical to cellular excitability. Maintenance of Ca²⁺ homeostasis is therefore crucial for the functions of both excitable and non-excitable cells. Interestingly, many TRP ion channels have evolved sophisticated auto-regulatory mechanisms allowing them to self-regulate Ca²⁺ entry for the tight control of Ca²⁺ fluctuations and downstream Ca²⁺ signaling. Understanding the Ca²⁺-dependent regulation of TRP ion channels is therefore not only essential for elucidating TRP channel mediated physiology and diseases, but also critical for understanding the Ca²⁺ signaling network.

Calmodulin (CaM), a founding member of Ca²⁺-binding proteins, is a fascinating Ca²⁺ signaling regulator. Firstly, CaM exhibits high sensitivity to Ca²⁺ conferred by the four EF-hand domains, a well-characterized Ca²⁺-binding motif. These EF-hand domains form two globular lobes (N- and C-lobe), with each lobe containing two EF-hand domains but with differential Ca²⁺-binding affinity (K_d for N-lobe, 10⁻⁶ M; K_d for C-lobe, 10⁻⁷ M) [14]. Two lobes of CaM act as two independent Ca²⁺ sensors for decoding different Ca²⁺ signals and are the mechanistic origins of the lobe-specific regulation of P/Q-type Ca²⁺ channels and TMEM16A Cl⁻ channels by different Ca²⁺ levels [15,16]. Secondly, CaM assumes various conformations endowed by two independent lobes and a long flexible linker connecting the two lobes, allowing CaM to bind to and regulate a myriad of target proteins [17], including ion channels, such as small-conductance K⁺ (SK) channels [18,19], KCNQ channels, cyclic nucleotide-gated channels and voltage-gated Na⁺ channels [20,21]. These unique properties of CaM suggest a broad role for CaM in the Ca²⁺-dependent regulation of TRP ion channels.

Indeed, a growing body of research has demonstrated the wide involvement of CaM in the Ca²⁺ regulation of TRP ion channels, including the TRPV, TRPM, TRPA1 and TRPC subfamily channels. These studies have substantially expanded the function of CaM and advanced our understanding of TRP channel modulation. Apart from CaM, Ca²⁺ modulates TRP channels by direct binding to TRP ion channels and by activating Ca²⁺-dependent enzymes. Here, we review the recent progress in the Ca²⁺ regulation of TRP ion channels, focusing on TRPV, TRPA, TRPM and TRPP channels. The Ca²⁺ regulation of TRPC channels is explained in the review by Zhu MX [22].

2. TRPV1

Capsaicin has traditionally been used as an analgesic for relieving pain. However, the analgesic mechanisms of capsaicin are poorly understood. The discovery of TRPV1 as a capsaicin receptor and its role in inflammatory pain suggested TRPV1 as the target of the analgesic actions of capsaicin [23,24,25]. It was soon found that persistent TRPV1 activation by capsaicin evokes channel desensitization and tachyphylaxis, a process that is dependent on Ca²⁺ and thought to underlie the analgesic effect of capsaicin [25,26]. However, how Ca²⁺ induces TRPV1 desensitization remains unclear.

Several studies have implicated a role for CaM in Ca²⁺-dependent desensitization of TRPV1, though there is a discrepancy regarding the precise regions in TRPV1 targeted by CaM [27,28,29]. A 35 amino acid peptide region in the distal C-terminus of TRPV1 was initially proposed as the CaM binding domain (CaMBD), because deletion of this region abolished CaM binding and blunted TRPV1 desensitization [27]. Consistently, this region was found to bind to two lobes of CaM with high affinity (K_d = 5.4 × 10⁻⁸ M) in an antiparallel orientation through hydrophobic and electrostatic interactions [30,31]. Soon after, a second CaMBD was identified overlapping with the first ankyrin repeat domain (ARD) in the N-terminus of TRPV1 (N-CaMBD) [28,29]. Interestingly, this region coincidently binds to ATP [29], which is known to enhance TRPV1 activity and prevent TRPV1 desensitization [32,33]. It was therefore proposed that activated Ca²⁺/CaM as a result of TRPV1 opening and Ca²⁺ influx binds to the ARD and displaces ATP, leading to channel inactivation and desensitization [29]. In further support of this idea, mutating the ATP binding sites on the ARD abolished Ca²⁺/CaM binding to the ARD and prevented Ca²⁺-dependent TRPV1 desensitization [29,30]. Although C-CaMBD is a high affinity Ca²⁺/CaM binding site, N-CaMBD appears to play a more important role in TRPV1 desensitization [30].

How do these two CaMBDs act in concert to mediate TRPV1 desensitization? An interesting possibility is that Ca²⁺/CaM binds to both N- and C-CaMBD through two independent lobes forming a tertiary complex, resulting in cross-linking or dimerization of two adjacent TRPV1 subunits, as seen in small conductance Ca²⁺-activated K⁺ (SK) channels [19]. This possibility is also supported by the resolved TRPV1 structure showing that the distal C-terminus of TRPV1 was in contact with the ARD from an adjacent TRPV1 subunit (Figure 1) [34]. However, this possibility was not demonstrated in size exclusion chromatography (SEC) through analysis of the binding of two TRPV1 CaMBD fragments [30]. Structural determination of TRPV1 in complex with Ca²⁺/CaM may be a better approach to resolve this possibility in the future.

In addition to a direct effect on TRPV1, Ca²⁺/CaM also activates a number of downstream kinases and phosphatases, such as protein phosphatase 2B (PP2B, calcineurin) [17]. A role for PP2B in the Ca²⁺-dependent desensitization of TRPV1 was demonstrated in experiments showing that cyclophilin, a specific PP2B inhibitor, prevents TRPV1 desensitization [35,36]. Interestingly, we further found that PP2B mediated TRPV1 desensitization depends on AKAP79/150, a scaffold protein anchoring PKA, PKC and PP2B in close proximity to TRPV1 through binding to the C-terminus of TRPV1 [37], because the Ca²⁺ desensitization of TRPV1 was reduced by either downregulating AKAP79, by specific deletion of the PP2B binding region on AKAP79, or by a peptide disrupting TRPV1–AKAP79 interaction [37]. However, this finding was not reproduced using calcium imaging [38]. It should be noted that the fluorescent dye used in calcium imaging binds to Ca²⁺, therefore buffering Ca²⁺-dependent downstream processes. Furthermore, calcium imaging is an indirect measurement of TRPV1 activity. In contrast, we used whole-cell patch clamping to directly record TRPV1 currents and selected cells with similar peak currents for experiments, as TRPV1 desensitization critically depends on the initial level of Ca²⁺ influx. These differences may account for the negative finding by Por et al. [38].

Not surprisingly, Ca²⁺-dependent desensitization of TRPV1 is subject to modulation by PKA and PKC, which are known to sensitize TRPV1 by enhancing TRPV1 phosphorylation [39,40,41]. Activation of either PKA or PKC reversed TRPV1 desensitization [36,39,42,43,44]. These data suggest that PP2B promotes TRPV1 desensitization by dephosphorylation of TRPV1, counteracting channel phosphorylation by protein kinases. However, it remains unknown whether PP2B dephosphorylates the same phosphorylation sites targeted by PKA and PKC.

In addition to Ca²⁺/CaM-dependent regulation of TRPV1 desensitization, considerable evidence also supports the involvement of membrane phosphatidylinositol-4,5-biphosphate (PIP₂). Firstly, PIP₂ has been shown to activate TRPV1 in excised patches [29,45,46,47]; secondly, Ca²⁺ influx during TRPV1 activation substantially decreases PIP₂ [48]; thirdly, Ca²⁺-dependent desensitization of TRPV1 can be prevented either by inhibition of PIP₂ degradation with a phospholipase C (PLC) inhibitor or by supplementing PIP₂ through the patch pipette [29,46]; and lastly, deletion of a Ca²⁺-sensitive PLC significantly has been shown to reduce TRPV1 desensitization [48]. These findings support a critical role for Ca²⁺-induced hydrolysis of PIP₂ in TRPV1 desensitization.

Based on these findings, we synthesize a model of Ca²⁺-induced desensitization of TRPV1. Upon TRPV1 opening and Ca²⁺ influx, Ca²⁺/CaM becomes activated and binds to both N- and C-CaMBD, inducing structural changes in TRPV1, leading to channel inactivation. The high affinity binding of Ca²⁺/CaM to C-CaMBD also activates nearby PP2B anchored on AKAP79/150 that binds to a nearby upstream region of C-CaMBD. PP2B then dephosphorylates TRPV1, counteracting channel phosphorylation by anchored PKA and PKC on AKAP79/150, promoting TRPV1 desensitization (Figure 2). Meanwhile, Ca²⁺ activates Ca²⁺-sensitive PLC, resulting in the cleavage of membrane PIP₂, further strengthening channel desensitization.

3. TRPV2

TRPV2 was initially characterized as a sensor for noxious heat, similar to TRPV1 [49]. However, mice lacking TRPV2 were shown to exhibit normal heat sensation [50], arguing against this initial hypothesis. Later on, it was found that TRPV2 is critical for macrophage phagocytosis and innate immunity [51], and is also involved in cancer migration and insulin secretion [52].

Like TRPV1, TRPV2 also exhibits Ca²⁺-dependent desensitization [53]. However, unlike TRPV1, the ARD in the N-terminus of TRPV2 does not bind to CaM or ATP [53,54]. Although an equivalent CaMBD has been identified in the proximal C-terminal region of TRPV2 [53,55], Ca²⁺/CaM binding did not play a role in Ca²⁺-induced TRPV2 desensitization [53]. Instead, desensitization of TRPV2 was found mediated by a concomitant reduction in the membrane PIP₂ as a result of Ca²⁺ entry through the channel, because Ca²⁺-dependent TRPV2 desensitization was reduced by dialyzing cells with soluble diC8-PIP₂ through the patch pipette [53]. Presumably, increased Ca²⁺ through TRPV2 preferentially activates a Ca²⁺-sensitive PLC, leading to cleavage of PIP₂. This is a possibility remaining to be investigated. The functional significance of TRPV2 desensitization is unclear, but it likely protects cells from the toxic effects of excessive Ca²⁺.

4. TRPV3

TRPV3 is also a thermosensitive ion channel activated by temperature ranges above 30–33 °C [56]. However, the role of TRPV3 in thermosensation in animals has not been consistently demonstrated [57,58,59]. An emerging theme is that TRPV3 plays a prominent role in skin physiology and diseases such as dermatitis [6,56].

A key feature of TRPV3 is that repetitive TRPV3 activation sensitizes its own responses, a process critically dependent on Ca²⁺ [60]. This Ca²⁺-dependent sensitization is in sharp contrast to Ca²⁺-induced desensitization of TRPV1 and TRPV2. Interestingly, sensitization of TRPV3 by Ca²⁺ is mediated by a conserved Ca²⁺/CaM and ATP binding site in the ARD in the N-terminus, analogous to that in TRPV1. In contrast to sensitization of TRPV1 by ATP, TRPV3 has been shown to be inhibited by ATP and also by Ca²⁺/CaM binding [54,60], which acts by shifting channel voltage dependence towards positive potentials [60]. This has been demonstrated by several approaches, including buffering increases of [Ca²⁺]_i, pharmacological inhibition of CaM, depletion of CaM with anti-CaM monoclonal antibody, and disrupting CaM binding through mutation, all of which potentiated TRPV3 currents, and reduced or abolished channel sensitization and voltage dependence [54,60]. It was therefore proposed that repetitive TRPV3 activation reduces the binding affinity of TRPV3 to Ca²⁺/CaM, leading to channel disinhibition and consequently channel sensitization. However, whether there is an actual reduction in Ca²⁺/CaM-TRPV3 binding during channel activation remains to be investigated.

A second mechanism for Ca²⁺-dependent modulation of TRPV3 arises from a direct Ca²⁺ block at a negatively charged residue, Asp641, in the pore region of the channel. Mutating this residue eliminated high affinity inhibition by Ca²⁺ (<10 μM), while sparing the effect of low affinity inhibition by Ca²⁺, which is mediated by Ca²⁺/CaM as described above [60]. TRPV3 therefore employs two distinct mechanisms to mediate channel sensitization by Ca²⁺, which is believed to underlie enhanced skin sensitivity to allergens and skin sensitizers [61].

5. TRPV4

TRPV4 is another member of thermosensitive TRP channels activated by low osmolarity and warm temperatures (≥27 °C) [3], and has been implicated in mechanical transduction, pain and itch [62]. Mutations in TRPV4 cause many genetic disorders, such as Charcot–Marie–Tooth disease and spinal muscular atrophy, due to dysregulated activation of TRPV4 [63].

Similar to other thermo-TRPs, Ca²⁺ also regulates TRPV4, but exerts a dual effect, initially promoting channel activation followed by channel inactivation [64]. Potentiation of TRPV4 can be induced by Ca²⁺ released from intracellular stores or by extracellular Ca²⁺ influx through the channel pore [64,65], suggesting that Ca²⁺ acts on intracellular channel sites. Consistently, two different CaMBDs were identified by two independent groups: one in the ARD in the N-terminus (N-CaMBD) (P132-I383) that also binds to ATP, analogous to that found in TRPV1 and TRPV3 [54]; and another in the C-terminus of TRPV4 (C-CaMBD, H812-E831) [65]. Disrupting CaM binding to C-CaMBD through mutation abolished Ca²⁺-induced potentiation of TRPV4 and significantly slowed down channel activation rate [65]. Mechanistically, it was initially proposed that Ca²⁺/CaM binding displaces and releases an inhibitory interdomain interaction with C-CaMBD from the N-terminus of TRPV4, similar to CaM-dependent activation of CaMKII [17]. This idea was supported by the finding that disrupting the interaction between the N-terminus and C-CaMBD enhanced TRPV4 currents, as did Ca²⁺/CaM, and also abolished Ca²⁺-induced channel potentiation [66]. However, a different autoinhibitory domain in the C-terminus (795–804), immediately upstream of C-CaMBD, was demonstrated to be critical. Firstly, deleting this domain resulted in constitutive channel activity and eliminated Ca²⁺/CaM modulation; and secondly, two gain-of-function TRPV4 mutants, E797K and P799L, underlying heritable skeletal dysplasia are also within the proposed autoinhibitory domain and exhibited reduced modulation by Ca²⁺/CaM [67]. Furthermore, C-CaMBD also mediates sensitization of TRPV4 by inositol triphosphate (IP₃), as deletion of C-CaMBD abolished IP₃ binding and also prevented sensitization of TRPV4 by IP₃ [68]. C-CaMBD may therefore be a converging point through which Ca²⁺ and other modulators regulate TRPV4 activation.

Although the role of C-CaMBD in Ca²⁺-dependent regulation of TRPV4 is clear, the role of N-CaMBD in this process remains elusive. Notably, N-CaMBD not only binds to ATP and CaM, but also directly binds to PIP₂ [69]. Functionally, TRPV4 is sensitized by ATP [54], but inhibited by PIP₂ [69]. The net effect of Ca²⁺/CaM binding to N-CaMBD is therefore uncertain. Ca²⁺/CaM could prevent ATP and/or PIP₂ from binding to N-CaMBD, causing either channel inactivation, as seen in TRPV1 [29], or channel potentiation. Interestingly, N-CaMBD (P132-I383) also overlaps with the initially proposed inhibitory domain (S117-K136) in the N-terminus [66]. Therefore, it is tempting to speculate that Ca²⁺/CaM binds to both N- and C-CaMBD through two different lobes forming a tertiary complex coupling Ca²⁺ signals to channel activation, as demonstrated for SK channels [18], though this possibility has not been supported by protein binding assay [66]. In general, Ca²⁺-induced TRPV4 potentiation has been well-studied. However, study on Ca²⁺-induced TRPV4 inactivation is still lacking. Whether it is mediated by Ca²⁺/CaM as suggested above remains an open question.

6. TRPV5 and TRPV6

TRPV5 is mainly expressed in epithelial cells in the kidney, playing a major role in renal Ca²⁺ reabsorption, whilst TRPV6 is expressed in intestine epithelial cells responsible for Ca²⁺ absorption [70]. Therefore, both TRPV5 and TRPV6 are critical for systemic Ca²⁺ homeostasis [70]. Consistent with their functional role, TRPV5 and TRPV6 are highly selective for Ca²⁺, acting as specialized epithelial Ca²⁺ channels and the gatekeepers of transcellular Ca²⁺ transport. This is in contrast to other TRP channels that are nonselective to cations.

The high Ca²⁺ selectivity is determined by a single aspartate residue (TRPV5-D542, TRPV6-D541) in the pore forming region, as mutation of these residues abolishes Ca²⁺ permeation of TRPV5 and TRPV6 [70,71]. Once opened, Ca²⁺ entry rapidly triggers inactivation of TRPV5 and TRPV6 with TRPV6 undergoing a much faster inactivation rate than TRPV5, providing a Ca²⁺-dependent feedback mechanism for preventing excessive Ca²⁺ influx [72].

Similar to other TRPV channels, CaM is responsible for Ca²⁺-induced inactivation of TRPV5 and TRPV6. For TRPV5, a distal C-terminal CaMBD (696–729) is crucial [73,74]. Mutating two residues, W702 and R706, in this region has been shown to diminish Ca²⁺-dependent channel inactivation [74]. An equivalent distal C-terminal CaMBD (695–727) was also found in TRPV6. Deleting this region not only abolished Ca²⁺-dependent interaction between TRPV6 and CaM, but also significantly blunted Ca²⁺-elicited channel inactivation [75,76]. Furthermore, overexpressed CaM, but not CaM₁₂₃₄, in which the four Ca²⁺-binding sites are mutated and therefore Ca²⁺-insensitive, exhibited a Ca²⁺-dependent interaction with TRPV6 and correspondingly reduced TRPV6 currents [75]. These data support a dynamic rather than a constitutive interaction of CaM with TRPV6 [75,77]. CaM binding regions were also identified in other regions of TRPV5 and TRPV6, including the N-terminus, transmembrane region and other parts of C-terminus [55,77,78]. However, Ca²⁺-dependent interaction with CaM was barely observed for some of the postulated CaMBDs in live cells [75], and importantly, no functional evidence is currently available supporting the involvement of these regions in Ca²⁺ inactivation of TRPV5 and TRPV6.

Interestingly, it has recently been reported that the two lobes of CaM bind to two separate subdomains within the distal C-CaMBD of TRPV6 in a 1:2 stoichiometry, with C-lobe constitutively binding to a region formed between S691 and L703 at low Ca²⁺, while high Ca²⁺ triggers the binding of N-lobe to a region between L707 and R708 [79]. A “two-tail” model has therefore been proposed. In accordance with this model, under resting low Ca²⁺, C-lobe CaM constitutively binds to the S691-L703 region through W695 on a C-tail of TRPV6. An elevation in [Ca²⁺]_i due to channel opening, loads Ca²⁺ onto N-lobe. Ca²⁺-loaded N-lobe then switches to bind to a second C-tail from another subunit leading to channel inactivation. Indeed, a TRPV6 mutant (L707A) enhanced N-lobe binding and also promoted TRPV6 inactivation [79]. N-lobe is therefore the determinant of channel inactivation. If this is the case, the N-lobe mutant CaM₁₂ deficient in Ca²⁺ binding should also abrogate Ca²⁺-dependent inactivation of TRPV6, which remains to be tested. Clearly, more structural and functional evidence will be required to validate this model.

Ca²⁺-induced inactivation of TRPV5 and TRPV6 is also modulated by other signaling pathways that interfere with CaM binding. For example, increased phosphorylation of C-CaMBD in TRPV5 induced by parathyroid hormone impaired CaM binding and therefore enhanced TRPV5 activity [74]. Similarly, phosphorylation of CaM binding region in TRPV6 by PKC also counteracted CaM binding and consequently slowed down channel inactivation [76].

Furthermore, a CaM-independent mechanism has also been suggested to be involved in TRPV6 inactivation [75]. This involves Ca²⁺-induced activation of PLC and subsequent hydrolysis of PIP₂, similar to that found in TRPV1 and TRPV2 channels. Indeed, PIP₂ has been found to be sufficient to activate TRPV6, and, moreover, Ca²⁺-evoked inactivation of TRPV6 has found to be reduced by either inhibiting PLC or by dialysis of PIP₂ into the cell [80,81]. Inactivation of TRPV6 caused by Ca²⁺ is therefore the collective action of both Ca²⁺/CaM and PIP₂ depletion [82].

7. TRPA1

TRPA1 channels detect diverse noxious thermal, chemical and mechanical stimuli, acting as a damage sensor and involved in various disease conditions, such as pain, itch, neuropathy and inflammation [3,10,83]. Ca²⁺ plays a significant role in the regulation of TRPA1. Firstly, Ca²⁺ directly activates TRPA1 and is essential for the basal responses of TRPA1 at lower Ca²⁺ concentrations (<1 mM) [84,85,86]. Ca²⁺ activation of TRPA1 may underlie diversified functions of TRPA1 channels. Indeed, Ca²⁺-dependent activation of TRPA1 has been proposed as a mechanism of TRPA1 activation by noxious cold [84]. Elevations in [Ca²⁺]_i by Gq-PLC-coupled receptors also activate TRPA1. TRPA1 may also be potentiated by Ca²⁺ influx through other nearby Ca²⁺-permeable channels, such as TRPV1, that form a complex with TRPA1 [87,88]. Secondly, Ca²⁺ inhibits basal TRPA1 responses and rapidly inactivates TRPA1 at higher Ca²⁺ concentrations (>1 mM) [86,89]. Ca²⁺ therefore exerts a dual and opposite effect on TRPA1 [86].

However, the mechanisms of Ca²⁺ regulation of TRPA1 have been controversial. Previous studies have suggested that the direct binding of Ca²⁺ to TRPA1 is responsible and have excluded the role of CaM in these processes, since dominant-negative CaM₁₂₃₄ was found to have no effect in Ca²⁺ regulation of TRPA1 [84,85,90]. On the contrary, we demonstrated that CaM is not only responsible for Ca²⁺-dependent potentiation (CDP) and inactivation (CDI) of TRPA1, but also determines the opposite effects of different Ca²⁺ levels on the basal responses of TRPA1 [86]. Importantly, TRPA1-CaM binding was Ca²⁺-dependent, and Ca²⁺-binding deficient CaM₁₂₃₄ did not bind to TRPA1 [86], which explains the lack of effects of CaM₁₂₃₄ in the regulation of TRPA1 observed previously [84]. We also identified a non-canonical CaMBD containing 17 amino acids in the C-terminus of TRPA1. Mutations with this region either abolished or diminished CDP and CDI [86]. However, it remains unclear how CaM mediates the opposite Ca²⁺ effects on TRPA1 and whether other regions of TRPA1 are also involved. In particular, the N-terminus of TRPA1 exhibited weak CaM binding and has been shown to be critical for Ca²⁺-induced desensitization of TRPA1 [86,91].

8. TRPM2

TRPM2 detects warm temperatures and is implicated in thermal sensation and regulation [92,93]. Notably, TRPM2 is also activated by reactive oxygen species (ROS) and ADP-ribose (ADPR) produced during oxidative stress and ischemia, functioning as a sensor for oxidative stress [9]. Ca²⁺ influx and Ca²⁺ signaling triggered by TRPM2 activation during oxidative stress is critical to many pathological processes such as cytokine production, cell death and immune and inflammatory diseases [9].

In addition to initiating downstream Ca²⁺ signaling, Ca²⁺ also activates TRPM2 and even TRPM2 splice variants that are insensitive to the channel agonist ADPR, suggesting that Ca²⁺ and ADPR open TRPM2 through distinct mechanisms [94]. Interestingly, Ca²⁺ enhanced ADPR-induced activation of TRPM2, but in the absence of Ca²⁺, ADPR was unable to activate TRPM2 [94,95]. Ca²⁺ therefore directly gates and modulates TRPM2 independently of ADPR [94,95,96]. Both Ca²⁺-dependent activation and modulation of TRPM2 are mediated by CaM, based on the following evidence: (1) dominant-negative CaM₁₂₃₄ inhibited TRPM2 activation; (2) CaM bound to an IQ-like motif domain in the N-terminus of TRPM2 in a Ca²⁺-dependent manner; (3) mutations in the IQ motif of TRPM2 impaired CaM binding and prevented TRPM2 activation by Ca²⁺ and ADPR [94,96]. Taken together, Ca²⁺ determines the activation and function of TRPM2.

A question remaining unclear is whether Ca²⁺-induced inactivation of TRPM2 [95] is mediated by CaM or by different Ca²⁺-interaction sites yet to be investigated.

9. TRPM3

TRPM3 is a sensor for noxious heat critical for heat nociception together with TRPV1 and TRPA1 [97,98]. TRPM3 is permeable to Ca²⁺ and constitutively active [99,100]. Ca²⁺ influx by TRPM3 was shown to be enhanced by depleting intracellular Ca²⁺ stores or by activating the Gq-coupled muscarinic receptor [100], suggesting the Ca²⁺-dependent activation of TRPM3. Interestingly, the N-terminus of TRPM3 contains two regions responsible for interaction with CaM and S100A1, two Ca²⁺-binding proteins [101]. However, neither Ca²⁺-dependent activation of TRPM3 nor the functional role of the postulated CaM binding domains has been directly investigated.

Interestingly, a recent study showed that activation of the Gq-coupled M1 muscarinic receptors rapidly inhibits TRPM3 through released Gβγ [102]. The direct inhibition of TRPM3 by Gβγ has also been reported by two other independent labs [103,104]. Activation of Gq-coupled receptors may therefore produce a mixed effect on TRPM3: an initial rapid inhibition by Gβγ followed by facilitation of activation of TRPM3 by Ca²⁺ due to concomitant activation of the Gq-PLC pathway. This possibility remains to be tested in the future.

10. TRPM4 and TRPM5

Both TRPM4 and TRPM5 channels are involved in the transduction of taste stimuli, such as sweet and bitter [105,106]. In contrast to other TRP channels, TRPM4 and TRPM5 are monovalent-selective and do not conduct divalent cations such as Ca²⁺ [107,108,109]. However, [Ca²⁺]_i is both necessary and sufficient for activation of both channels [107,109]. As anticipated, TRPM4 and TRPM5 are also activated by the Gq-protein coupled receptors that cause increases in [Ca²⁺]_i [106,107,108,109,110,111].

TRPM5 was shown to be activated by [Ca²⁺]_i between 0.3 μM and 1 μM, similar to TRPM4, but inhibited at higher [Ca²⁺]_i (≥1 μM) [107], which is in contrast to TRPM4, which exhibits no significant inhibition at higher Ca²⁺ levels [109]. Furthermore, activation of TRPM5 by [Ca²⁺]_i was rapidly followed by pronounced channel inactivation or desensitization, leading to the distinctive transient activation of the channel [107], which is different from more persistent activation of TRPM4 [109]. The rapid inactivation of TRPM5 was partially reversed by PIP₂ and was suggested to be Ca²⁺-dependent [110,112], though it was also proposed as a Ca²⁺-independent process [107]. TRPM4 currents undergo a similar rapid channel rundown or desensitization, a process attributed to the hydrolysis of PIP₂, a positive regulator of TRPM4 [111,113]. As Ca²⁺ entry through other TRP channels (e.g., TRPV1, TRPV2 and TRPV6, see above) has been shown to be sufficient to induce PIP₂ hydrolysis, it is therefore likely that PIP₂ hydrolysis, as a consequence of Ca²⁺-triggered activation of PLC, contributes to the desensitization of TRPM4 and TRPM5.

Two other different mechanisms are also involved in the Ca²⁺ sensitivity of TRPM4. The first mechanism involves CaM, similar to other TRP channels. This is evidenced by the finding that Ca²⁺ activation of TRPM4 is markedly decreased by co-expressing the dominant negative CaM₁₂₃₄ and that direct application of CaM to excised TRPM4 channels prevents TRPM4 desensitization [114]. In an attempt to identify CaMBD on TRPM4, several CaM binding sites in the N- and C-termini of TRPM4 were found. Consistently, deletion of C-terminal CaM binding sites impaired Ca²⁺ activation of TRPM4 [114]. The second mechanism is mediated by the direct binding of Ca²⁺ to two negatively charged residues (D1049 and E1062) near and in the TRP domain. Neutralization of these residues reduced Ca²⁺ sensitivity [115]. However, a recently resolved TRPV4 structure revealed that the Ca²⁺ binding site is within the intracellular side of the S1–S4 domain [116]. For TRPM5, Ca²⁺ sensitivity is the direct action of Ca²⁺ on TRPM5, independently of CaM, because Ca²⁺ is sufficient to induce TRPM5 activity in excised patches and CaM inhibitors have been shown to have no effect on the Ca²⁺ activation of TRPM5 [108,110]. However, the exact Ca²⁺ binding sites on TRPM5 have not yet been identified.

11. TRPM8

TRPM8 channels are activated by cold temperatures (≤30 °C) and cooling compounds, such as menthol, acting as a cold sensor [117] and setting it apart from the warm- or heat-sensitive TRPM2–5 channels [92,97,118].

TRPM8 is permeable to Ca²⁺, and Ca²⁺ influx upon channel opening triggers rapid channel desensitization [119], a process thought to underlie cold adaptation. In the absence of Ca²⁺, TRPM8 currents are substantially larger and lack desensitization [119]. It was thought that Ca²⁺ acts by mainly activating Ca²⁺-sensitive PLCδ4, leading to hydrolysis of PIP₂, a regulator essential for maintaining channel activity [120,121,122,123]. Consistently, TRPM8 currents were shown to be larger and to exhibit reduced desensitization in PLCδ4-deficient sensory neurons [123]. However, the significant desensitization of TRPM8 was still observed in PLCδ4-lacking neurons [123], suggesting other unknown mechanisms that also underlie Ca²⁺-dependent desensitization of TRPM8. Nevertheless, PLCδ4-lacking mice exhibited increased duration and events of TRPM8-dependent nocifensive behaviors in response to cooling [123]. Similarly, pharmacologically blocking PLC also inhibited the responses of paw withdrawal of the mice to cooling [124]. These experiments suggest that PLC-mediated hydrolysis of PIP₂ underlies TRPM8 desensitization and cold adaptation in vivo.

12. TRPP2

TRPP2 is also known as polycystin-2 (PC2) or PKD2 and is permeable to Ca²⁺ [125]. Mutations in TRPP2 is a cause of autosomal dominant polycystic kidney disease (ADPKD), a consequence thought to be due to aberrant calcium signaling of the mutated channel [126]. Understanding the interaction between TRPP2 and Ca²⁺ is therefore important for elucidating the mechanisms of ADPKD.

Ca²⁺ exerts a dual effect on TRPP2 with lower concentrations of Ca²⁺ (≤0.3 μM) facilitating, and higher concentrations of Ca²⁺ inhibiting, the channel, leading to a bell-shaped Ca²⁺ dependence [127]. The Ca²⁺ sensitivity of TRPP2 channels is believed to be due to a direct binding of Ca²⁺ to the channel. Indeed, a single Ca²⁺ binding site was revealed by NMR in the EF-hand domain (720–797) in the C terminus of TRPP2 [128]. Mutation of the EF-hand domain abolished Ca²⁺ binding and rendered the mutated channel inactive [129,130], suggesting that the EF hand domain is a Ca²⁺ sensor. On the other hand, phosphorylation of TRPP2 at S812 close to the EF-hand domain by casein kinase II (CK2) increased the Ca²⁺ sensitivity of TRPP channels, because mutation of S812 markedly reduced Ca²⁺-dependent activation and inactivation of TRPP2, leading to a positive shift in Ca²⁺-dependence [127]. Presumably, phosphorylation affects the binding affinity of Ca²⁺ to the EF hand domain. However, the single Ca²⁺ binding site in the EF hand domain cannot account for the dual effects of Ca²⁺ on TRPP2. Therefore, Ca²⁺-dependent regulation of TRPP2 likely involves other unknown mechanisms.

13. Concluding Remarks

Spatial and temporal control of Ca²⁺ signaling is necessary for many physiological processes. TRP ion channels are not only the major contributors to Ca²⁺ signaling, but also are the targets of Ca²⁺ signaling, ensuring rapid control of functions of TRP ion channels and associated Ca²⁺ signaling. Ca²⁺-regulation of TRP ion channels largely involves three major mechanisms: direct binding of Ca²⁺ to channels, Ca²⁺-sensing CaM and Ca²⁺-dependent hydrolysis of PIP₂. Of them, CaM is probably the most common mechanism in mediating Ca²⁺-dependent regulation of TRP ion channels. CaM not only directly senses different levels of Ca²⁺ through two different lobes, but also serves as a multifunctional effector protein, providing vast versatility for decoding Ca²⁺ signals and modulating TRP ion channels. Future research is required to further understand diversified regulation of TRP ion channels by CaM. Accumulating evidence also suggests the presence of other Ca²⁺-dependent mechanisms critical in the regulation of TRP ion channels, which remain to be investigated.