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Cannabinoids are a major class of compounds produced by the plant Cannabis sativa. Previous work has demonstrated that the main cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC) can have some beneficial effects on pain, inflammation, epilepsy, and chemotherapy-induced nausea and vomiting. While CBD and THC represent the two major plant cannabinoids, some hemp varieties with enzymatic deficiencies produce mainly cannabigerolic acid (CBGA). We recently reported that CBGA has a potent inhibitory effect on both Store-Operated Calcium Entry (SOCE) via inhibition of Calcium Release-Activated Calcium (CRAC) channels as well as currents carried by the channel-kinase TRPM7. Importantly, CBGA prevented kidney damage and suppressed mRNA expression of inflammatory cytokines through inhibition of these mechanisms in an acute nephropathic mouse model. In the present study, we investigate the most common major and minor cannabinoids to determine their potential efficacy on TRPM7 channel function. We find that approximately half of the tested cannabinoids suppress TRPM7 currents to some degree, with CBGA having the strongest inhibitory effect on TRPM7. We determined that the CBGA-mediated inhibition of TRPM7 requires a functional kinase domain, is sensitized by both intracellular Mg⋅ATP and free Mg2+ and reduced by increases in intracellular Ca2+. Finally, we demonstrate that CBGA inhibits native TRPM7 channels in a B lymphocyte cell line. In conclusion, we demonstrate that CBGA is the most potent cannabinoid in suppressing TRPM7 activity and possesses therapeutic potential for diseases in which TRPM7 is known to play an important role such as cancer, stroke, and kidney disease. Graphical Abstract [ABSTRACT FROM AUTHOR]

Store-operated [Ca.sup.2+] entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER [Ca.sup.2+] sensors with calcium release-activated calcium (CRAC) channels (CRACM1/2/3 or Orail/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20-amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels. 10.1085/jgp.201010484

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane [Ca.sup.2+]-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide ([H.sub.2][O.sub.2]) evokes [Ca.sup.2+] influx through TRPM2 to activate [Ca.sup.2+]-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-[kappa]B essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, [H.sub.2][O.sub.2]-induced [Ca.sup.2+] influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 [Ca.sup.2+] influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases., The biological purpose of inflammation is to bring fluids, proteins and inflammatory cells such as neutrophils and monocytes from the blood into the damaged tissues to eliminate the injuring agents [...]

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), [Ca.sup.2+] and calmodulin (CAM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as [Cs.sup.+] or [Na.sup.+] can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular [Ca.sup.2+] as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using [K.sup.+]-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of [Ca.sup.2+], both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular [Ca.sup.2+], a minimum of 30 nM internal [Ca.sup.2+] is required to cause partial TRPM2 activation with ADPR. However, 200 [micro]M external [Ca.sup.2+] is as efficient as 1 mM [Ca.sup.2+] in TRPM2 activation, indicating an external [Ca.sup.2+] binding site important for proper channel function. [Ca.sup.2+] facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular [Ca.sup.2+]. Furthermore, TRPM2 currents inactivate if intracellular [Ca.sup.2+] levels fall below 100 nM irrespective of extracellular [Ca.sup.2+]. The facilitatory effect of intracellular [Ca.sup.2+] is not mimicked by [Mg.sup.2+], [Ba.sup.2+], or [Zn.sup.2+]. Only [Sr.sup.2+] facilitates TRPM2 as effectively as [Ca.sup.2+], but this is due to [Sr.sup.2+]-induced [Ca.sup.2+] release from internal stores rather than a direct effect of [Sr.sup.2+] itself. Together, these data demonstrate that cytosolic [Ca.sup.2+] regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 [micro] M CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [[[Ca.sup.2+]].sub.i] and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and [Ca.sup.2+] facilitates activation via calmodulin.

TRPM7 is a [Ca.sup.2+]- and [Mg.sup.2+]-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular [Mg.sup.2+], the functional roles of intracellular levels of Mg x ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg x ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free[ Mg.sup.2+]. Mg x ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible [Mg.sup.2+] and [Ca.sup.2+] overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP [greater than or equal to] GTP [greater than or equal to] UTP > ITP [approximately equal to] free [Mgsup.2+] alone. These nucleotides also enhanced TRPM7 inhibition by free [Mg.sup.2+], suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the [Mg.sup.2+]-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg x NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible [Mg.sup.2+] binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a [Mg.sup.2+] binding site extrinsic to that domain.

Significant progress in the molecular and functional characterization of a subfamily of genes that encode melastatin-related transient receptor potential (TRPM) cation channels has been made during the past few years. This subgroup of the TRP superfamily of ion channels contains eight mammalian members and has isoforms in most eukaryotic organisms. The individual members of the TRPM subfamily have specific expression patterns and ion selectivity, and their specific gating and regulatory mechanisms are tailored to integrate multiple signaling pathways. The diverse functional properties of these channels have a profound effect on the regulation of ion homoeostasis by mediating direct influx of [Ca.sup.2+], controlling [Mg.sup.2+] entry, and determining the potential of the cell membrane. TRPM channels are involved in several physiological and pathological conditions in electrically excitable and non-excitable cells, which make them exciting targets for drug discovery.

Transient receptor potential (TRP) proteins are a diverse family of proteins with structural features typical of ion channels. TRPM5, a member of the TRPM subfamily, plays an important role in taste receptors, although its activation mechanism remains controversial and its function in signal transduction is unknown. Here we characterize the functional properties of heterologously expressed human TRPM5 in HEK-293 cells. TRPM5 displays characteristics of a calcium-activated, nonselective cation channel with a unitary conductance of 25 pS. TRPM5 is a monovalent-specific, nonselective cation channel that carries [Na.sup.+], [K.sup.+], and [Cs.sup.+] ions equally well, but not [Ca.sup.2+] ions. It is directly activated by [[[Ca.sup.2+]].sub.i] at concentrations of 0.3-1 [micro]M, whereas higher concentrations are inhibitory, resulting in a bell-shaped dose-response curve. It activates and deactivates rapidly even during sustained elevations in [[[Ca.sup.2+]].sub.i], thereby inducing a transient membrane depolarization. TRPM5 does not simply mirror levels of [[[Ca.sup.2+]].sub.i], but instead responds to the rate of change in [[[Ca.sup.2+]].sub.i] in that it requires rapid changes in [[[Ca.sup.2+]].sub.i] to generate significant whole-cell currents, whereas slow elevations in [[[Ca.sup.2+]].sub.i] to equivalent levels are ineffective. Moreover, we demonstrate that TRPM5 is not limited to taste signal transduction, because we detect the presence of TRPM5 in a variety of tissues and we identify endogenous TRPM5-like currents in a pancreatic beta cell line. TRPM5 can be activated physiologically by inositol 1,4,5-trisphosphate-producing receptor agonists, and it may therefore couple intracellular [Ca.sup.2+] release to electrical activity and subsequent cellular responses.

Trace metal ions such as [Zn.sup.2+], [Fe.sup.2+], [Cu.sup.2+], [Mn.sup.2+], and [Co.sup.2+] are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a [Ca.sup.2+]- and [Mg.sup.2+]-permeable cation channel, whose activity is regulated by intracellular [Mg.sup.2+] and [Mg.sup.2+]*ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for [Zn.sup.2+] and [Ni.sup.2+], which both permeate TRPM7 up to four times better than [Ca.sup.2+]. Similarly, native MagNuM currents are also able to support [Zn.sup.2+] entry. Furthermore, TRPM7 allows other essential metals such as [Mn.sup.2+] and [Co.sup.2+] to permeate, and permits significant entry of nonphysiologic or toxic metals such as [Cd.sup.2+], [Ba.sup.2+], and [Sr.sup.2+]. Equimolar replacement studies substituting 10 mM [Ca.sup.2+] with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of [Zn.sup.2+] [approximately equal to] [Ni.sup.2+] >> [Ba.sup.2+] > [Co.sup.2+] > [Mg.sup.2+] [greater than or equal to] [Mn.sup.2+] [greater than or equal to] [Sr.sup.2+] [greater than or equal to] [Cd.sup.2+] [greater than or equal to] [Ca.sup.2+], while trivalent ions such as [La.sup.3+] and [Gd.sup.3+] are not measurably permeable. With the exception of [Mg.sup.2+], which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with [Mn.sup.2+], [Co.sup.2+], or [Ni.sup.2+] suggest that these can be transported by TRPM7 in the presence of physiological levels of [Ca.sup.2+] and [Mg.sup.2+], suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells. KEY WORDS: zinc * magnesium * nucleotide * trp channel * cation channel

Results reveal that the 'transient receptor potential' TRPM4b exhibits calcium-activated nonselective channel properties, which is activated by calcium. However, TRPM4b does not carry calcium but depolarizes cells in a calcium-dependent manner, suggesting that TRPM4b is a calcium-activated nonselective channel involved in cell depolarization.

Author(s): Meredith C. Hermosura [1]; Hiroshi Takeuchi [2]; Andrea Fleig [1]; Andrew M. Riley [3]; Barry V. L. Potter [3]; Masato Hirata [2]; Reinhold Penner (corresponding author) [1] Receptor-mediated generation [...]