Ca²⁺-activated nonselective cation channels were first described in the early 1980s [1,15,31], shortly after the patch-clamp recording technique was developed [5]. Patch-clamp recording, and in particular the excised-patch recording configuration, made it possible to rapidly and reproducibly change the intracellular milieu of cells and therefore to discover intracellular activators and modulators of ion channels. In this context, intracellular Ca²⁺ at micromolar concentrations was found to activate ion channels that were equally permeable to Na⁺ and K⁺ ions, and these ion channels were detected in a variety of cell types. The Ca²⁺-activated nonselective channels found in cultured heart cells were proposed to play a role in modulating the duration of the action potential, whereas those in pancreatic acinar cells were proposed to play a role in exocrine secretion [1,15]. However, without specific pharmacological or genetic tools, a clear attribution of function was difficult. In 2002 this situation changed with the cloning and functional expression of the first Ca²⁺-activated non-selective cation channel, TRPM4 [9]. Following this discovery, a second Ca²⁺-activated cation channel, TRPM5, was characterized [6,12,22,24,32]. Our understanding of the biological significance of these channels, their functional regulation, and their structural properties has since proceeded at a rapid pace.

CLONING, DISTRIBUTION, AND STRUCTURE OF TRPM4 AND TRPM5

TRPM4 and TRPM5 are members of the TRPM subfamily of transient receptor potential ion channels. The first member of the TRPM subfamily of ion channels, melastatin (TRPM1), was identified as a gene that was downregulated in mouse melanoma cell lines [2]. Based on similarity to TRPM1, a partial sequence of TRPM4 was detected in the database of human-expressed sequence tags and used to identify full-length cDNAs with two possible splice variants encoding proteins of 1,040 (TRPM4a) [30] and 1,214 (TRPM4b) [9] amino acids. Subsequently, only TRPM4b has been shown to be functional and therefore the term TRPM4 will be used to refer to this splice variant. TRPM4’s message is expressed widely and is at its highest levels in the heart, prostate, colon [30], placenta, and pancreas [9]. No localization of the mRNA or protein has yet been reported within these tissues.

TRPM5 was initially identified by homology to TRPM1 [4,23] and was later found by two independent groups to be a key component of mammalian taste cells [22,32]. Presently only a single splice variant has been reported. In contrast to TRPM4, TRPM5 mRNA is expressed in a highly restricted manner, and among major tissues of the body it is found only at high levels in the tongue, small intestine, and stomach [22]. Lower levels of TRPM5 may be expressed in other tissues [23].

TRPM4 and TRPM5, which are structurally more related to each other than to other TRP channels, show ~40 percent amino acid identity. Based on hydropathy analysis and homology to voltage-activated ion channels, both have been proposed to contain six transmembrane-spanning regions and to assemble as tetramers. The cytoplasmic N- and C-termini of TRPM4 and TRPM5 contain a number of potential protein–protein binding sites: the C-termini of both channels contain predicted coiled-coil domains and N- and C-termini of TRPM4 contain calmodulin-binding sites [19]. The pore of TRPM4 appears to be located between the fifth and sixth transmembrane domains, an assignment that is supported by the observation that mutations within this region alter ion selectivity [17]. Moreover, a histidine residue in the C-terminal portion of this region mediates an external proton block of TRPM5, supporting the contention that this region forms the outer pore vestibule [13].

ION PERMEABILITY, UNITARY PROPERTIES, AND SPECIFIC BLOCKERS OF TRPM4 AND TRPM5

Both TRPM4 and TRPM5 are robustly activated by intracellular Ca²⁺, making it possible to study their function properties in detail with patch-clamp recording [6,9,12,16,24]. These studies have revealed a number of similarities and differences between the two channels that will be helpful in classifying native channels and in understanding structure-function relationships. Both channels have a unitary conductance of ~25 pS [6,9,12,24], but whereas the openings of TRPM4 channels are long-lived (several hundred milliseconds; e.g., see reference 33), openings of TRPM5 channels are transient (e.g., see reference 12). Both channels are permeable to monovalent cations and impermeable to divalent cations [6,9,12,24].

At present only a few “specific” blockers of TRPM4 and TRPM5 channels have been identified. TRPM4 channels are blocked by micromolar concentrations of intracellular ATP and other adenine nucleotides [20], whereas TRPM5 channels are insensitive to these small molecules. TRPM5 channels are blocked by external pH of 6.0 or lower [13], whereas TRPM4 channels are insensitive to acid pH.

Based on these characteristics, one can assign the likely genetic basis for native channel activity; for example, based on the above criteria, the Ca²⁺-activated channels expressed in pheromone-sensing cells of the vomeronasal organs, which show long openings and are blocked by adenine nucleotides, are likely encoded by TRPM4 [10].

SENSITIVITY OF TRPM4 AND TRPM5 TO INTRACELLULAR CA²⁺

One of the most important ways in which TRPM4 and TRPM5 channels differ is in sensitivity to intracellular Ca²⁺. In excised inside-out patches immediately upon patch excision, the EC₅₀ for activation by Ca²⁺ of mTRPM4 is 100–200 μM [27,33], and the EC₅₀ for activation by Ca²⁺ of mTRPM5 is 20–30 μM [12,27] (Figure 15.1). Sensitivity to Ca²⁺ decreases for both channels by ~four-fold following patch excision and exposure to Ca²⁺ ; the EC₅₀ for activation by Ca²⁺ after desensitization is ~500 μM and ~80 μM for TRPM4 and TRPM5, respectively [12,33]. Somewhat mysteriously, TRPM4 and TRPM5 currents in whole-cell recording mode are ~10–50 times more sensitive to Ca²⁺, and both can be maximally activated by concentrations of Ca²⁺ as low as 1 μM (e.g., see reference 27). It is not known why there is such a large discrepancy in Ca²⁺ sensitivity between excised-patch and whole-cell recording modes. One possibility is that a factor is lost upon patch excision that controls sensitivity (see below).

FIGURE 15.1

Ca²⁺-dependent activation of TRPM4 and TRPM5 currents. (A,B) Responses of TRPM4 and TRPM5 currents to increasing concentrations of Ca²⁺. The lower panels show currents recorded from the same patches after desensitization. (C) Dose-response relations of (more...)

ACTIVATION OF TRPM4 AND TRPM5 BY VOLTAGE

In the presence of an activating concentration of intracellular Ca²⁺, depolarization strongly increases the opening of TRPM4 and TRPM5 channels [6,12,16,26]. This is apparent in the strong outward rectification of TRPM4 and TRPM5 currents in response to voltage ramps, despite the fact that the underlying channels display a linear I–V relation [6,12,16,24,26]. In response to depolarizing voltage steps, both TRPM4 and TRPM5 currents show time-dependent relaxation, reflecting the opening of channels (Figure 15.2B). Voltage-dependent activation has also been reported for TRPM8 and TRPM4 and thus may be a common feature of TRPM channels [6,16,21,25]. It has been hypothesized that the weak voltage dependence of these channels allows their gating to be easily modulated [21], a hypothesis that is supported by work on cold regulation of TRPM8, TRPV1, and TRPM5 [26,29], decavanadate modulation of TRPM4 [18], and PI(4,5)P₂ regulation of TRPM4 and TRPM8 [25,33] (see below). The structural mechanism of voltage sensing of these TRP channels is not presently understood, but the presence of several positively charged residues in the fourth transmembrane of these channels suggests that this may be the voltage sensor [7,21].

FIGURE 15.2

TRPM5 currents are Ca²⁺ and voltage dependent. (A) In whole-cell recording mode, 40 μM Ca²⁺ in the pipette elicited a large rectifying current in a HEK293 cell expressing TRPM5. Recording began shortly after break in to the whole-cell mode. Inset (more...)

REGULATION OF TRPM4 AND TRPM5 BY PIP₂

A consistent observation is that TRPM4 and TRPM5 currents run down following activation, regardless of whether the currents are measured in whole-cell, perforated-patch, or excised-patch recording modes (Figure 15.2A and Figure 15.3). In excised patches, this rundown is associated with a decrease in the Ca²⁺ sensitivity of the channels, and thus can be formally considered desensitization [12,33]. What is the basis for desensitization? Recent evidence indicates that it is at least in part due to hydrolysis of membrane phophoinosides, most likely by Ca²⁺-activated membrane-associated PLCs [28]. The evidence for this conclusion includes the demonstration that (1) both TRPM5 and TRPM4 currents can be rescued from desensitization by PI(4,5)P₂ [12,33] (Figure 15.3C); (2) TRPM4 currents can be rescued from desensitization by intracellular MgATP at concentrations that activate lipid kinases and are expected to restore PI(4,5)P₂ levels, and this effect can be blocked by lipid kinase inhibitors [19,33] (Figure 15.3A); (3) desensitization of TRPM4 is promoted by depletion of PI(4,5)P₂ with polyamines (Figure 15.3B).

FIGURE 15.3

ATP and PI(4,5)P₂ restore TRPM4 currents from desensitization. Inward currents evoked in response to 100 μM cytoplasmic Ca²⁺ were recorded from inside-out patches from TRPM4-expressing ChoK1 cells (Vm = −80 mV). (A) Following desensitization, (more...)

At present the mechanism and structural determinants for PI(4,5)P₂ regulation of TRPM4 and TRPM5 is not known. Detailed study shows that PI(4,5)P₂ changes the voltage-dependent gating of TRPM4 channels, promoting channel activation at negative membrane potentials by stabilizing the channels in the “voltage-activated” regulation state [33]. This mechanism is similar to one proposed to explain PI(4,5)P₂ regulation of the structurally related, cold-activated channel TRPM8 [25]. Sensitivity to PI(4,5)P₂ is conferred by positively charged residues in the TRP domain of TRPM8, a loosely conserved sequence of 25 amino acids located adjacent to the sixth transmembrane domain [25]. It is not known whether this region also mediates interaction of TRPM4 with PI(4,5)P_2.

FUNCTION OF TRPM4 AND TRPM5

Insight into the function of TRPM5 came with the discovery that its expression is largely restricted to taste cells and the GI tract [22,32]. Taste consists of five distinct modalities of which three—bitter, sweet, and umami—are mediated by G-protein-coupled receptors. These receptors activate a signaling cascade of which several downstream targets have been identified, including Gα13, gustducin, and PLCβ2 [11,14]. TRPM5 is coexpressed with all three types of G-protein-coupled receptors as well as with their downstream signaling components [22,32], suggesting that it acts in the pathway for bitter, sweet, and umami taste transduction. In support of this idea, knockout of TRPM5 or PLCβ2 severely impairs the ability of mice to detect bitter, sweet, and umami tastes [32]. Based on the present genetic and physiological data, a preliminary model for transduction of bitter, sweet, and amino acid tastes can be proposed. In this model, receptor activation of PLCβ2 generates IP₃, which releases Ca²⁺ from intracellular stores, and store-released Ca²⁺ opens TRPM5 channels. Opening of TRPM5 channels leads to a depolarization of the taste cell and transmitter release. Adaptation of taste responses may be partly mediated by the hydrolysis of PI(4,5)P₂, which is a cofactor for activation of TRPM5 [12] (Figure 15.4, left).

FIGURE 15.4

Models for physiological activation of TRPM5 and TRPM4. (Left) A model for taste transduction. Binding of taste stimuli to G-protein-coupled taste receptors (T1R and T2R) leads to dissociation of the heterotrimeric G protein. βγ subunits (more...)

TRPM4’s function has been more difficult to discern and a knockout of TRPM4, which would expedite this task, has not yet been reported. Moreover, TRPM4 mRNA is widely distributed, suggesting that it may play a less specific functional role than TRPM5. Despite these difficulties, there is strong evidence that TRPM4 plays a role in cytokine secretion by T lymphocytes and myogenic constriction of cerebral arteries [3,8]. T lymphocytes respond to stimulation with phyohemagglutinin (PHA) with Ca²⁺ oscillations that release the cytokine IL-2. Dominant negative suppression of TRPM4 and RNA knockdown of TRPM4 prolongs the Ca²⁺ response, leading to an increase in IL-2 production [8]. Thus TRPM4 appears to play a role in dampening the response of T cells to PHA. Based on these results, a model has been proposed in which activation of T-cell receptors induces a PLC-signing pathway leading to the depletion of Ca²⁺ from intracellular stores and the consequent entry of Ca²⁺ through CRAC channels. Ca²⁺ entry triggers activation of Ca²⁺-activated K⁺ channels, the opening of which promotes entry of Ca²⁺ by increasing the driving force across the plasma membrane. Activation of TRPM4 by Ca²⁺ entering through CRAC channels causes a depolarization of the membrane potential, which opposes further Ca²⁺ entry and promotes the opening of V-gated K⁺ channels. The interplay of these four conductances generates the Ca²⁺ oscillations observed in response to stimulation of T-cell receptors [8] (Figure 15.4, right).

Two models for the functional roles of Ca²⁺-activated TRPs have been proposed [8,12]. One posits that TRPM5 is activated by Ca²⁺ released from intracellular stores and that TRPM5 generates the primary electrical response to tastants. The second posits that TRPM4 is activated by Ca²⁺ entry through CRAC channels and that the primary function of TRPM4 is to dampen this signaling pathway. In this context, it may be significant that TRPM4 and TRPM5 differ widely in Ca²⁺ sensitivity and thus may respond to different Ca²⁺ signals in cells. For example, the higher sensitivity of TRPM5 to Ca²⁺ may allow it to respond to global changes in Ca²⁺ concentration following release of Ca²⁺ from intracellular stores, whereas TRPM4 might be insensitive to such signals and respond only to entry of Ca²⁺ through closely opposed membrane Ca²⁺ channels. A challenge will be to determine the sources of Ca²⁺ that activate these two channels under physiological conditions and to identify the specific attributes of each channel that allow it to respond selectively to the appropriate stimulus.

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After this chapter was submitted, Nilius et al. [34] showed a similar regulation of TRPM4 by PIP₂ as we described.