Transient receptor potential, subfamily M(melastatin-related), member 4 (TRPM4) gene encodes a channel protein responsible for a Ca2+-activated nonselective cationic (NSCCa) current. Electrophysiological signatures of such currents are known since the beginning of patch clamp single-channel recordings. They were observed in a wide variety of tissues in the 1980s to 2000s, including epithelia, secretory tissues, or excitable cells (neurons and myocytes) (see Guinamard et al. 2011 for review). However, these currents remained orphaned until the cloning of the TRP gene family at the end of the 1990s. Among these, TRPM1 (melastatin) has been cloned in 1998 and opened the way for the new subfamily TRPM which is composed of eight members (1–8). Based on homology sequence screening of a cDNA library, Xu et al. identified a first sequence of the TRPM4 gene in human (Xu et al. 2001) encoding for a 1040 amino acid protein. Later, a longer splice variant (1214 amino acids) has been cloned (Launay et al. 2002) followed by a shorter one (677 amino acids) (Nilius et al. 2003). These splice variants are successively designated as TRPM4a, TRPM4b, and TRPM4c. However, while TRPM4b protein appeared later as the molecular determinant of most of the NSCCa currents, the physiological significance of TRPM4a and TRPM4c are still in debate. According to this, TRPM4 will be used in the following sections to refer to the full-length protein TRPM4b.
Identification of the TRPM4 protein provided the opportunity to unravel the physiological and pathological implications of the TRPM4 channel. Generation of Trpm4 null mice or RNA interference (iRNA) gave the opportunity to reveal the effect of gene disruption (Barbet et al. 2008; Vennekens et al. 2007). This was concomitant with the description of TRPM4 inhibitors (Ullrich et al. 2005; Grand et al. 2008). In addition, the discovery of TRPM4 mutations associated with cardiac dysfunctions revealed its implication in human physiology and physiopathology (Kruse et al. 2009; Guinamard et al. 2015 for review). These contemporary studies conducted in the 2010s provided a portrait of the roles of TRPM4 in a large variety of processes such as immune response, insulin secretion, smooth muscle activity, onset of respiratory activity, cardiac electrical and mechanical activity, cell differentiation or migration, and ischemia or stroke-induced central nervous system damages.
TRPM4 Structure and Expression Profile
TRPM4 mRNA has been detected widely among tissues. In human, it has been reported at high expression level in the heart, pancreas, placenta, and prostate but at a lower level in the kidney, skeletal muscles, liver, intestine, thymus, and spleen (Launay et al. 2002; Nilius et al. 2003). It has also been detected in hematopoietic cell lines including T and B lymphocytes (Launay et al. 2002). At the protein level, TRPM4 is actually considered as mostly expressed at the plasma membrane rather than membranes from organelles. Note that glycosylation of TRPM4 is necessary to stabilize channel expression at the plasma membrane. In addition, phosphorylation influences basolateral targeting in epithelial cells.
TRPM4 Current Properties and Pharmacology
Since TRPM4 is an ion channel, its physiological relevance derives from its ionic current properties. TRPM4 is permeable to monovalent cations, mainly Na+ and K+ that it does not differentiate. By contrast, it is not permeable to Ca2+, unlike most of other TRP channels (Launay et al. 2002, Nilius et al. 2003). Permeability sequence is thus Na+ = K+ > Cs+ > Li+ >> Ca2+. In symmetrical ionic conditions, the single-channel current exhibits a linear current-voltage relationship with a conductance of 20–25 pS (Launay et al. 2002; Guinamard et al. 2011 for review). While it is not considered as a canonic voltage-gated channel, its activity increases with membrane depolarization. This results in a typical outward rectification of the whole-cell current. Channel open probability is also finely regulated by internal Ca2+ concentration. Ca2+ activates the channel with a concentration for half efficiency in the range of few μmol.L−1 (Ullrich et al. 2005). TRPM4 is inhibited by internal adenosine nucleotides (ATP, ADP, AMP) with a concentration for half maximal inhibition in the range of the μmol.L−1 (Ullrich et al. 2005; Mathar et al. 2014 for review). Note that this point is a major discrepancy with the TRPM5 channel which also produces an NSCCa current but which is not sensitive to adenosine nucleotides (Ullrich et al. 2005).
Beside of these major regulations, TRPM4 current has also been shown to be activated by phosphatidylinositol 4,5-bisphosphate (PIP2) which uncouple channel activity from voltage variations and increases sensitivity to Ca2+. PKC and oxygen species such as H2O2 also increase TRPM4 activity (for reviews, see Mathar et al. 2014; Guinamard et al. 2011).
Identification of TRPM4 roles in physiology requires pharmacological modulators. However TRPM4 still lacks specific modulators. It is inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid with a concentration for half maximal inhibition, IC50 = 3 × 10−6 mol.L−1. Albeit flufenamic acid is known to target a large range of other ion channels, TRPM4 is among the more sensitive. Spermine and kinin also inhibit the channel (Ullrich et al. 2005). Interestingly, the antidiabetic sulfonylurea glibenclamide inhibits TRPM4 (for reviews, see Mathar et al. 2014; Guinamard et al. 2011), a property which opens potential clinical applications as described in the section named “TRPM4 as a New Drug Target for Drug Design”. To date, the most specific inhibitor is the hydroxytricyclic compound 9-phenanthrol (IC50 = 2 × 10−5 mol.L−1) (Grand et al. 2008).
TRPM4 Partners Proteins
TRPM4 has been shown to co-localize and physically bind with TRPC3 in HEK-293T transfected cells (Cho et al. 2015 for review). While such association generates a current which properties do not strictly match those of TRPM4 or TRPC3, it is not clear whether the complex is an association of the two proteins or a heteromerization of TRPM4 and TRPC3 in a single heterotetramer. Moreover, the association remains to be confirmed in native cells. Nevertheless, it opens the possibility that TRPM4 associates with other TRP isoforms to form specific channels.
It has also been shown that TRPM4 heteromerizes with the sulfonylurea receptor 1 (SUR1). The complex confers to TRPM4 a higher sensitivity to ATP and glibenclamide and increases its affinity for Ca2+ and calmodulin. The TRPM4-SUR1 complex inhibition has been shown to reduce neuroinflammation in subarachnoid hemorrhage in human, paving the way for glibenclamide use to protect against brain injuries following ischemic stroke (see Caffes et al. 2015 for review).
Partner proteins have also been shown to modulate TRPM4 trafficking. The binding protein 14-3-3γ association with TRPM4 at the N-terminus increases forward trafficking to the plasma membrane, as shown in HEK-293T cells and in HT22 cells, a phenomenon involved in glutamate-induced neuronal cell death (Cho et al. 2015 for review).
TRPM4 may also associate with small ubiquitin-like modifier (SUMO) family members. Channel deSUMOylation is involved in channel reinternalization after expression to the plasma membrane. A mutation in the N-terminus (E7K) has been identified in a patient with cardiac conduction block and shown to be responsible for increased SUMOylation and thus decrease in channel reinternalization (Kruse et al. 2009). Such endocytosis is also affected by other mutations (R164W, A432T, G844D) identified in additional patients with cardiac conduction blocks (see Guinamard et al. 2015 for review) and associated to deregulation of SUMOylation.
TRPM4 Physiological Significance
The wide mRNA detection within tissues (Launay et al. 2002; Nilius et al. 2003) and the functional recording of TRPM4-like currents in most of the cells where it has been looked for let suspect a large spectrum of physiological implications. Indeed, TRPM4 has been shown, since 15 years, to be involved in numerous processes. Given the wide range of implication of TRPM4, we will refer, in the following lines, to specific reviews to drive the reader.
TRPM4 in Immune Cells
TRPM4 in Heart
Heart expresses a high level of TRPM4 mRNA (Launay et al. 2002) with the greatest expression in the conductive tissue, moderate in the atria, and the lowest in the ventricle (see Guinamard et al. 2015 for review). The same gradient has been observed for functional TRPM4 currents. Physiologically, TRPM4 has been shown to support the pacemaker activity of the sinus node cells (Fig. 3b) and prolong Purkinje fibers as well as atrial and ventricular cardiomyocyte action potentials. It is also involved in cardiac dysfunctions since inherited TRPM4 mutations have been found in families with members showing cardiac conduction blocks or Brugada syndrome (Fig. 1) (Kruse et al. 2009; Guinamard et al. 2015 for review). Most of these mutants lead to a variation (gain or loss) in TRPM4 current density at the plasma membrane when heterologously expressed in HEK-293 cells. These variations are due to modifications of TRPM4 protein expression at the plasma membrane, while single-channel electrophysiological properties are not affected. However, the mechanistic by which channel mutation leads to the specific cardiac disease is not well understood, and current modifications remain to be confirmed in cardiomyocytes. TRPM4 also participates in hypoxia-reoxygenation-induced arrhythmias. Finally TRPM4 might also be involved in the control of cardiac hypertrophy since TRPM4 knockout mice develop such hypertrophy due to hyperplasia (Guinamard et al. 2015 for review).
TRPM4 in Insulin Secretion by Pancreatic β-Cells
In addition to other channels, TRPM4 modulates insulin secretion by pancreatic β-cells from the islets of Langerhans (Fig. 3c). In those, TRPM4, activated by cytosolic Ca2+ increase or the PLC/PKC pathway, would depolarize the cells, producing an activation of voltage-gated Ca2+ channels. Such activation increases Ca2+ entry necessary for the fusion of internal vesicles with the plasma membrane and thus insulin secretion (see Islam 2011 for review; Shigeto et al. 2015). In this signaling, TRPM4 might be a link between Gq-coupled receptors which activates the PLC/PKC pathway and insulin secretion, as it has been shown for the vasopressin-induced insulin secretion.
TRPM4 in Smooth Muscle Cells
TRPM4 modulates the activity of a variety of smooth muscle myocytes (see Earley 2013 for review). In those cells, Gq-coupled receptor activation leads to IP3 receptor opening and Ca2+ release from sarcoplasmic reticulum which activates TRPM4. Na+ entry through TRPM4 produces a depolarization which opens the voltage-dependent Ca2+ channel (VDCA) allowing Ca2+ entry initiating cell contraction. This has been first observed in cerebral arteries (Fig. 3d) where TRPM4 acts as a bridge in the mechanosensation that leads to vasoconstriction according to the Bayliss effect. More recently, TRPM4 has been shown to promote the contraction of the detrusor smooth muscle of the urinary bladder (Hristov et al. 2016). Finally, TRPM4 is also expressed in colonic smooth muscle, but its physiological implication remains to be evaluated (see Earley 2013 for review).
TRPM4 in Neurons
TRPM4 participates in neuronal activity. In that field, TRPM4 implication has been mostly investigated in the context of the onset of breathing activity by pacemaker neurons from the pre-Bötzinger complex located in the brainstem (see Funk 2013 for review). In this region, an inspiratory neuronal network involves glutamatergic transmission through AMPA receptors and metabotropic receptors (Fig. 3e). Gq-coupled metabotropic receptors activate the PLC/IP3 pathway, leading to intracellular Ca2+ stores release and TRPM4 activation. The resulting TRPM4-depolarizing current enhances bursting activity of pacemaker neurons, in combination with other depolarizing currents induced by the voltage-dependent Ca2+ channels and AMPA receptors.
TRPM4 as a New Target for Drug Design
Since it is involved in a large variety of physiological processes and their malfunctions, TRPM4 is now considered as an interesting drug target in medicine. However, little is known about the feasibility of its modulation in the aim to correct such malfunctions. An increasing work has been done recently on central nervous system injuries and their treatments by glibenclamide which is known to inhibit the SUR1-TRPM4 complex (see Caffes et al. 2015 for review). The complex is overexpressed in neurons, astrocytes, oligodendrocytes, and vascular endothelial cells in focal ischemia and hemorrhage. In these pathologies, Ca2+ overload results in cell death. Thus SUR1-TRPM4 expression may be beneficial in a first step since the resulting current decreases Ca2+ overload by reducing the electrochemical driving force for Ca2+ entry. However, it becomes noxious with time since Na+ entry through TRPM4 leads to Na+ overload followed by cellular edema which may terminate in cell membrane rupture, as occurs in necrosis. According to this, pharmacological inhibition of SUR1-TRPM4 by glibenclamide was hypothesized to have neuroprotective properties. This point is still under consideration, but promising results have already been obtained in human in the context of ischemic and hemorrhagic stroke (Sheth et al. 2016).
According to the complex but strong contribution of TRPM4 in cardiac physiology and dysfunctions, including arrhythmias (see Guinamard et al. 2015 for review) as well as in vascular functions, the channel has also to be considered as a valuable target in cardiovascular disease correction. However, to date, no clinical data are available.
Transient receptor potential, subfamily M(melastatin-related), member 4 (TRPM4) gene encodes a channel protein with the classical molecular structure of TRP channels. TRPM4 mRNA, protein, and current have been detected in a large range of tissues, including excitable and non-excitable cells in human. TRPM4 channel is permeable to monovalent cations (Na+ and K+) but not Ca2+. It is activated by cytosolic Ca2+, PIP2, protein kinase C, and membrane depolarization but inhibited by internal ATP. According to these properties, TRPM4 opening leads to a depolarizing current due to Na+ entry in most of the cells. Thereby, it modulates the activity of voltage-dependent ion channels and the electrochemical driving force for ions. Since it is activated by cytosolic Ca2+, TRPM4 is a driving belt in internal Ca2+ signaling by transforming cytosolic Ca2+ variations into voltage variations.
A number of physiological roles have been described for TRPM4. In the immune system, it reduces immune factors secretion by T lymphocytes and influences dendritic cells migration. In the heart, it participates in the induction of pacemaker activity in the sinus node cells and prolongs action potentials from cardiomyocytes. It also enhances the onset of breathing activity by pacemaker neurons from the pre-Bötzinger complex located in the brainstem. TRPM4 influences insulin secretion by pancreatic β-cells. Finally, TRPM4 modulates the activity of a variety of smooth muscle myocytes including those from the vasculature and the detrusor muscle from the bladder.
TRPM4 has also been shown to be involved in pathologies. TRPM4 gene mutations leading to either loss or gain of function were found in patients with cardiac conduction blocks and Brugada syndrome. In addition, TRPM4, in combination with the sulfonylurea receptor type 1, promotes brain injuries in the context of ischemia. According to these implications, TRPM4 is now considered as a new promising target in cardiac or neuronal protection as well as in other tissues damages. A first therapeutic study targeting TRPM4 by pharmacological modulators is on the way in the context of neuroprotection.