Ca²⁺: Calcium; CMR1: Cold and Menthol Receptor 1; CRAC: Ca²⁺ Release-Activated Ca²⁺ channels; Cryo-EM: Cryo-Electron Microscopy; Cs+: Cesium; ER: Endoplasmic Reticulum; GPCR: G Protein-Coupled Receptor; ICRAC: Ca²⁺ Release-Activated Ca²⁺ Currents; INa: Sodium Currents; K+: Potassium; MCU: Mitochondrial Ca²⁺ Uniporter; Na⁺: Sodium; NCKX: K+-dependent Na⁺/Ca²⁺ Exchangers; NCLX: Na⁺- Li⁺/Ca²⁺ Exchangers; NCX: Na⁺/Ca²⁺ Exchangers; NHE: Na⁺/H+ Exchangers; PIP₂: Phosphatidylinositol 4,5-bisphosphate; Pirt: Phosphoinositide interacting regulator of TRP; SOCE: Store-Operated Ca²⁺ Entry; STIM1/2: Stromal Interaction Molecules 1-2; Thap: Thapsigargin; TRPM8: Transient Receptors Potential (TRP) Melastatin member 8

The electrochemical driving forces across the plasma membrane mediated by ion channels, pumps, and exchangers are essential for cellular homeostasis, regulating a wide range of biological processes [1,2]. Although both excitable (e.g., neurons) and non-excitable (e.g., lymphocytes) cells manage their cellular functions through plasmalemmal ion flux, excitable cells change the membrane potential mediated by depolarization and voltage-gated ion channels, while nonexcitable cells control this process by the different downstream processes and ligand-gated ion channels [2,3]. Sodium (Na⁺) is the principal extracellular cation, being carried to the intracellular space mainly through inward Na⁺ currents (INa) [2]. Pioneering studies documented that inhibition of INa, but not the calcium (Ca²⁺) absence, abolished the action potential, indicating that Na⁺ influx is essential for cell excitability, action potential, and cellular functionality [4,5]. Additionally, the Na⁺ driving forces also have been related to the function of several other ion transmembrane modes of transport, including 1) pumps (Na⁺/K+ ATPases); 2) Na⁺/Ca²⁺ exchangers (NCX/NCKX); 3) Na⁺/H+ exchangers (NHE); 4) ligand-dependent channels (nicotinic acetylcholine receptors); and recently linked to the 5) transient receptors potential (TRP) melastatin member 8 (TRPM8), which are permeable both to the Na⁺ and Ca²⁺ ion influx [2,6,7].

It’s well established that Ca²⁺ handling is one of the most widespread transducing systems, regulating a wide range of cell biology ranging from cell fertilization and hormone exocytosis to cell death and hypomineralization during pathophysiological processes [8-10]. Ca²⁺ release-activated Ca²⁺ (CRAC) channels are mediated by the endoplasmic reticulum (ER) resident Ca²⁺ sensors stromal interaction molecules 1-2 (STIM1/2) and by a highly Ca²⁺-selective conductance pore, known as ORAI (ORAI1-3) channels [11,12]. As shown in Figure 1, the ER-Ca²⁺ depletion elicits the Ca²⁺ influx through ORAI1-3 in many cells, including HEK-293, and both proteins orchestrally form the main store-operated Ca²⁺ entry denominated as SOCE. Ca²⁺ influx via SOCE represents the major Ca²⁺ entry pathway into a variety of non-excitable cells [11,13]. From a physiological viewpoint, ORAI1-3 channels are highly selective for Ca²⁺, being 1000-fold more conductive for Ca²⁺ ions than Na⁺ ions [14-16]. However, during experimental or pathological conditions when divalent cations are suppressed from the extracellular solution, ORAI1- 3 channels become permeable to monovalent cations as Na⁺, cesium (Cs+), or potassium (K+) [14,16].

Since the term, TRP was introduced and mammalian TRP channels identified, studies suggested that TRP homologs might represent the molecular components of SOCE and CRAC currents (ICRAC) [17-19]. Most TRP channels are nonselective cation channels widely expressed as cellular sensors with diverse physiological functions, being permeable to both monovalent and divalent cations with Ca²⁺/Na⁺ conductance ranging from 0.01 to 100 [20,21]. The elementary structure of TRP consists of six transmembrane helical domains (TM1- TM6) with a loop between TM5 and TM6 forming the channel pore and N- and C terminal regions located in the cytosol [21,22]. Some of the 28 mammalian members such as the TRPM7 channels contain a catalytic kinase domain and Nand/ or C-terminal coiled-coil, responsible for a key role in TRP multimerization and interaction with STIM and ORAI proteins [20]. Recent studies reported that some clustered members from the TRP subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin), and TRPN (NOMPC-like) are components of the SOCE [17,23]. However, subsequent reports failed to reproduce these outcomes [17-19]. On the other hand, Rosado’s laboratory demonstrated that although TRPC channels did not generate inward Ca²⁺ currents that resemble ICRAC, TRPC1 and other TRPC proteins might play a relevant role in modulating ORAI1 and STIM1 channel functionality [17,19,24]. Additionally, TRPM7 channels potentiate Ca²⁺ influx via SOCE, but its function is fully dependent on the prior activation of the ORAI1 channels [25,26]. Thus, despite this being beyond the scope of the present study, the controversy surrounding the role of TRP channels as part of or mediating SOCE stems from how intertwined these pathways are. In this review, the author aimed at the modulatory crosstalk between Na⁺ and Ca²⁺ signaling mediated by TRPM8 channels and SOCE components (ORAI1-3 and STIM1/2), and how these pathways affect other Na⁺ and Ca²⁺ ion transmembrane transports.

TRPM8 was originally cloned by screening cDNA isolated from trigeminal sensory neurons for their ability to respond to menthol and cold stimuli [27,28], although these channels are widely distributed in different tissues. TRPM8 can be expressed in somatosensory neurons, mucosae, urogenital tract, lung cells, and artery myocytes, which might be related to the allodynia and/or hyperalgesia process [7]. In the last years, the modulation of TRPM8 channels has been considered a prominent approach to develop a novel therapeutic tool for chronic pain and noxious cold sensitization [29]. The significance of these findings and the discovery of the touchsensitive Piezo1-2 channels [30] won the discoverers D. Julius and A. Patapoutian the 2021 Nobel Prize in Physiology [27,31]. Physiologically, there are two populations of thermal coldsensitive cells activated in temperature around 12°C, being the largest (≥ 70%) subpopulation sensitive to the TRPM8- ligand, menthol, that is a cold-sensitive compound [20,32,33]. Experimental studies using animals lacking TRPM8 channels revealed a deficient capacity to detect cool temperatures and exhibit a partially defective phenotype although the sensitivity to low temperature (~5 °C) induced cold pain remained intact in these TRPM8 knockout mice [7,34]. These outcomes suggest an important role of TRPM8 in the cold sensation probably associated with activation of other TRP channels and/or ion signaling pathways.

It is well established that TRPM8, a non-selective Ca²⁺- permeable channel, exhibits multi-gating mechanisms, being activated by innocuous cool to cold temperatures and regulated by crucial molecules such as Ca²⁺ ion and phosphatidylinositol 4,5-bisphosphate (PIP₂) [7]. The C-terminally located PIP₂ sensing domain is one determinant of temperature sensitivity [7,20]. Also, the intracellular Ca²⁺ is required for TRPM8 activation by cooling agonist, icilin, but not by menthol being mediated by residues in S2-3 domains [33,35]. TRPM8 channels are permeable to Na⁺ [36] with the ratio permeability between Ca²⁺ and Na⁺ ions (PCa/PNa) range between 0.97 to 3.2, with monovalent ions conductance of Cs+ > K+ > Na⁺ [7]. Also, these channels can depolarize the cells and activate voltage-gated Na⁺ and Ca²⁺ channels [37], leading to an increase in ions influx. Basal cytosolic Ca²⁺ is required for TRPM8 activation by the cooling agonist, icilin, but not for menthol activity [37]. Cryo-electron microscopy (Cryo-EM) structure studies revealed that the Ca²⁺-binding site in TRPM8 is conserved within the TRPM subfamily [20,38]. Additionally, the Y745 residue interacts with cooling agonists (icilin and WS-12) and antagonists (AMTB and TC-I 2014), suggesting the key role of Y745 in the ligand-dependent gating of TRPM8 [20,35,37]. Another modulator of TRPM8 channels is the PIP₂ that is required as a cofactor for channel activation by cooling agonists and cold temperatures [7,37,39]. Thus, at high concentrations, PIP₂ itself might be sufficient to activate the TRPM8 channel, whereas depletion of basal PIP₂ levels results in channel desensitization [37,40]. Endogenous ligands including testosterone, artemin, and Pirt (phosphoinositide interacting regulator of TRP) protein have been also proposed as physiological ligands of TRPM8 channels [7,29].

Recently, basic and clinical studies highlighted the TRPM8 channels as a novel pharmacotherapeutic target for the treatment of cancer, neuropathic pain, and inflammation [29,41]. Voltage is a partial activator of TRPM8 channels with low (<1.0) open probability values, decreasing when the temperature rises [36,37]. Electrophysiology data showed that the half-activation voltage (V0.5) saturates at high temperatures, suggesting a lack of strict coupling between TRPM8 voltage and temperature activation [36,37]. Also, TPRM8 channel deactivation at negative voltages and temperature can underestimate the open probability of these channels, showing that temperature affects channel opening through a pathway that does not involve voltage sensor activation [37]. Clinical outcomes have demonstrated that TRPM8 channels are involved in the modulation of cancer hallmarks such as cellular proliferation, survival, and invasion [42], suggesting that TRPM8 channels play contributory roles in tumor growth and metastasis. Several studies revealed that both TRPM8 and SOCE components (ORAI1-3 and STIM1/2) are upregulated in a variety of human neoplastic tissues, cell lines, and solid tumors [41-44]. These findings links TRPM8 and SOCE as well as Na⁺ and Ca²⁺ signaling during cancer. Thus, opening a new pathway to the development of the TRPM8 pharmacological modulator and as a prognostic/predictive biomarker and a therapeutic target in cancer treatment.

The sensory nervous system enables fine coordinated properties of TRP in a heterologous of varied external sensorial stimulus [20]. Although the molecular mechanism underlying their temperature sensitivity remains unclear, the TRP channels are essential to body thermoreceptors [21]. Different subfamilies of TRP channels are requested by regulating a wide temperature oscillation, including the TRPV1-2 in the painful heat, TRPV3-4 in non-painful warmth, and TRPA1 during noxious cold [45]. TRPM8 channels could be modulated by exogenous and endogenous ligands, being the cold-sensitive compounds derived from plants as menthol important TRPM8- ligand [33,35]. These channels also had been named as the cold and menthol receptor 1 (CMR1) being the main detector of cold in humans that is activated during innocuous cool to cold temperatures [7]. They are voltage-sensitive channels and can be activated by changes the membrane potential or depolarization, however, lack the voltage sensor present in the classic voltage-gated ion channels [7,37].

Recent Cryo-EM studies revealed that both Ca²⁺ ion and the phospholipid, PIP₂, have binding sites into transmembrane helical domains, and are required for the properly gating and activation of TRPM8 channels [15,33,37]. As shown in Figure 2 and previously reported [33,35,40], the cold temperature (~14-16°C) and the well-known cooling agents, icilin and menthol, can increase the [Ca²⁺]cyt levels around 100-800 nM range. Additionally, electrophysiological records reported that menthol triggers inward INa in trigeminal neurons and HEK-293 cells overexpressing TRPM8 channels [28,46]. Taken together, these outcomes indicate that TRPM8 can uptake Na⁺ and Ca²⁺ ions, increasing the Ca²⁺/Na⁺ cytosolic levels at the micromolar range. The permeability ratio between Ca²⁺ and Na⁺ ions (PCa/ PNa) range between 0.97 to 3.2 [7,32,37]. TRPM8 channels also can depolarize and activate voltage-gated Na⁺ and Ca²⁺ channels [37], leading to more Na⁺ and Ca²⁺ influx into the cytosol. This makes the TRPM8 the important molecule that integrates Na⁺/Ca²⁺ signaling pathways.

The modulation between Na⁺/Ca²⁺ ion uptake and intracellular cation signaling is finely regulated by transmembrane Na⁺/Ca²⁺ exchangers and pumps, and also can be buffered by organelles and cytosolic proteins [47,48]. Electrochemical gradients carry inward Na⁺ and Ca²⁺ currents that increase the cytosolic levels of both ions around 1-3 μM for Ca²⁺ and ≥ 40 mM for Na⁺ ions, cell-typing dependent [48-50]. Although organelles such as mitochondria and ER buffer these ions, the capacity to store and/or buffer Na⁺ and Ca²⁺ is limited due to the intracellular location and saturation of the buffer mechanisms [51,52]. For example, the mitochondria can uptake Ca²⁺ through the mitochondrial Ca²⁺ uniporter (MCU), and also stimulate the Na⁺ buffer mediated by NCLX (Na⁺-Li⁺/ Ca²⁺) exchangers [8,52,53]. By contrast, the ER is the largest Ca²⁺ intracellular organelle, can store Ca²⁺ in a range between 100 to 800 nM [54]. Thus, despite these intracellular buffers being essential for ion homeostasis, the plasma membrane low-affinity Na⁺/Ca²⁺ extrusion mechanisms appear to be important players and links between Na⁺ and Ca²⁺ signaling and TRPM8 channels.

The Na⁺/Ca²⁺ exchanger is a key electrogenic system that directly integrates opposite fluxes of Na⁺ and Ca²⁺ across the plasma membrane [50]. These Na⁺/Ca²⁺ exchangers are divided in NCX1-3 members encoded by SLC8A1-3 genes and in the K+-dependent Na⁺/Ca²⁺ exchangers (NCKX1-6) encoded by SLC24A1-6 being the K+ ions absolute requirement for Ca²⁺ clearance mediated by NCKX1-6 [55,56]. Both NCX and NCKX have been characterized by singular features that includes: 1) low Ca²⁺ affinity (>1 μM) and high Ca²⁺ capacity (~ 2500 s-1) responsible for the fast Ca²⁺ clearance; 2) NCX extrude 1 Ca²⁺ and uptake 3 Na⁺ and NCKX extrude 1 Ca²⁺ and 1 K+, and uptake 4 Na⁺; and 3) increase in cytosolic Na⁺ promote its function in the reverse mode in order to clearance excess of Na⁺ ions [55,57,58]. These complex feedback functions involve both thermodynamic and kinetic interactions of Na⁺ and Ca²⁺ with ion transport and regulatory domains of NCX1-3/NCKX1- 6 [55,56,58].

Although the molecular mechanisms responsible for the link between the TRPM8 and SOCE remain unknown, recently emerged some downstream pathways associated with these connections. The potential candidate proteins involved in this crosslink were investigated by in silico gene-protein prediction analyses targeting genes responsible for encoded TRPM8, ORAI1-3, STIM1/2, PLCβ, PIP₂, and Na⁺/Ca²⁺ exchanger (NCX1- 3/NCKX1-6). The in silico data revealed a predicted crosslink between these pathways-genes and TRPM8 channels (Figure 3), highlighting possible interconnectivity. TRPM8 activity can be altered by intracellular Ca²⁺ concentrations and PIP₂ levels, temperature, and pH, whereas endogenous signaling molecules modulate TRPM8-mediated responses to coolness [37]. In pulmonary smooth muscle cells, the TRPM8 agonist icilin caused inhibition of SOCE and a molecular modulation of TRPM8 channels may affect the pulmonary hypertension development related to SOCE activity [59]. Additionally, Zhang demonstrated that both the G protein-coupled receptor (GPCR) Gα_q-subunit and PIP₂ were able to inhibit TRPM8 channels, and the TRPM8-Gα_q binding impaired Gα_q coupling and signaling to PLCβ-PIP₂ [60]. In this way, multivariate analysis showed that overexpression of TRPM8, and STIM2 is associated with a lower risk of systemic recurrence after radical prostatectomy [61]. Taken together, these studies suggest that Gα_q-subunit and PLCβ-PIP₂ generating IP3 could be a novel insight to further understand the structure-function relationships of TRPM8 and SOCE. Another evidence of modulatory crosstalk between Na⁺ and Ca²⁺ signaling mediated by TRPM8 channels, SOCE, and Na⁺/Ca²⁺ exchanger is the teeth-related tissues responsible for the mineralization and pain processes [62,63]. Both animals and humans lacking or with loss-of-function mutation of the SOCE components (ORAI1/STIM1) or NCKX4 exchangers result in Amelogenesis Imperfecta and deficient enamel formation [10,56,64,65]. Also, the ability of TRPM8 to uptake Na⁺ and Ca²⁺ can affect the SOCE and NCKX4 functions, resulting in the disruption of Na⁺ and Ca²⁺ signaling during pathological conditions. TRPM8 has also been detected in isolated human odontoblasts and although TRPM8 knockout mice do not lack sensation to noxious cold, they exhibit reduced cold hypersensitivity following nerve injury and inflammation [34,66,67]. Therefore, this evidence makes TRPM8 channels, SOCE, and Na⁺/Ca²⁺ exchanger possible key targets molecule integrating Na⁺ and Ca²⁺ signaling pathways.

In numerous cell types control their downstream cascades pathways through a transmembrane fine balance between influx and efflux of Ca²⁺ and Na⁺ ions [68]. Compared to the Ca²⁺ signaling, the role of Na⁺ signaling in the cellular functions emerging now as a prominent target related to channelopathies [50,69,70]. New insights highlight TRPM8 channels as a possible player responsible for the link between Na⁺ and Ca²⁺ signaling [2,7,29,50]. Although no direct interaction between TRPM8, SOCE and Na⁺/Ca²⁺ exchangers function have been shown, the data reviewed above and the illustrative crosslink on Figure 4 suggest that TRPM8 could be the channel that links Na⁺ and Ca²⁺ signaling, providing a novel insight for further understanding of structure-function relationships of these cellular signaling pathways during the ionic influx and efflux. The development of new drugs or repurposing of well-known cooling agonists/antagonists has been challenged by the pharmacological profile of these compounds, being the TRPM8 channels a new target to be explored in the future.