ORAI Calcium Channels: Regulation, Function, Pharmacology, and Therapeutic Targets.

The changes in intracellular free calcium (Ca2+) levels are one of the most widely regulators of cell function; therefore, calcium as a universal intracellular mediator is involved in very important human diseases and disorders. In many cells, Ca2+ inflow is mediated by store-operated calcium channels, and it is recognized that the store-operated calcium entry (SOCE) is mediated by the two partners: the pore-forming proteins Orai (Orai1-3) and the calcium store sensor, stromal interaction molecule (STIM1-2). Importantly, the Orai/STIM channels are involved in crucial cell signalling processes such as growth factors, neurotransmitters, and cytokines via interaction with protein tyrosine kinase coupled receptors and G protein-coupled receptors. Therefore, in recent years, the issue of Orai/STIM channels as a drug target in human diseases has received considerable attention. This review summarizes and highlights our current knowledge of the Orai/STIM channels in human diseases and disorders, including immunodeficiency, myopathy, tubular aggregate, Stormorken syndrome, York platelet syndrome, cardiovascular and metabolic disorders, and cancers, as well as suggesting these channels as drug targets for pharmacological therapeutic intervention. Moreover, this work will also focus on the pharmacological modulators of Orai/STIM channel complexes. Together, our thoughtful of the biology and physiology of the Orai/STIM channels have grown remarkably during the past three decades, and the next important milestone in the field of store-operated calcium entry will be to identify potent and selective small molecules as a therapeutic agent with the purpose to target human diseases and disorders for patient benefit.

Strategies to Inspire Students' Engagement in Pharmacology Courses.

Pharmacology is a distinct discipline and offers core knowledge to broaden student programs in the provision of health care (medicine, nursing, pharmacy, and others) as well as research-oriented programs (biosciences and biomedical). Therefore, knowledge and information on topics such as prescribing medication, drug interactions, dosage adjustments, and the correct drug dose calculation for medicine are essential for preventing and reducing medication errors, which is a key element in patient safety. Recently, many institutions have been trying to enhance their quality of teaching, as students demand support and success in their studies. Pharmacology is a highly challenging interdisciplinary topic, and requires a huge effort from both educators and students to achieve the best outcomes. Therefore, creating an effective environment to support students' learning in pharmacology courses is essential to improving students' engagement, success and learning outcomes. In recent decades, the landscape of education has changed, and distance learning has skyrocketed. This manuscript intends to discuss and highlight the importance of student engagement in higher education in pharmacology courses. Additionally, this paper spotlights and presents a review of recent studies focusing on student engagement in pharmacology courses and possible methods for enhancing and inspiring student engagement in pharmacology courses.

Kir6.2-D323 and SUR2A-Q1336: an intersubunit interaction pairing for allosteric information transfer in the KATP channel complex.

ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to modulators was restored when Kir6.2-D323K was co-expressed with a reciprocal charge swap mutant, SUR-Q1336E. Importantly, equivalent interactions have been identified in both Kir6.1 and Kir6.2 suggesting this is a second important interaction between Kir6 and the proximal C terminus of SUR.

Mibefradil, a T-type Ca2+ channel blocker also blocks Orai channels by action at the extracellular surface.

BACKGROUND AND PURPOSE: Mibefradil, a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca2+ channels. EXPERIMENTAL APPROACH: Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS: Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 µM respectively. Outside-out patch demonstrated that perfusion of 10-µM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 µM) inhibited Ca2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity.

Treasure troves of pharmacological tools to study transient receptor potential canonical 1/4/5 channels.

Canonical or classical transient receptor potential 4 and 5 proteins (TRPC4 and TRPC5) assemble as homomers or heteromerize with TRPC1 protein to form functional nonselective cationic channels with high calcium permeability. These channel complexes, TRPC1/4/5, are widely expressed in nervous and cardiovascular systems, also in other human tissues and cell types. It is debatable that TRPC1 protein is able to form a functional ion channel on its own. A recent explosion of molecular information about TRPC1/4/5 has emerged including knowledge of their distribution, function, and regulation suggesting these three members of the TRPC subfamily of TRP channels play crucial roles in human physiology and pathology. Therefore, these ion channels represent potential drug targets for cancer, epilepsy, anxiety, pain, and cardiac remodelling. In recent years, a number of highly selective small-molecule modulators of TRPC1/4/5 channels have been identified as being potent with improved pharmacological properties. This review will focus on recent remarkable small-molecule agonists: (-)-englerin A and tonantzitlolone and antagonists: Pico145 and HC7090, of TPRC1/4/5 channels. In addition, this work highlights other recently identified modulators of these channels such as the benzothiadiazine derivative, riluzole, ML204, clemizole, and AC1903. Together, these treasure troves of agonists and antagonists of TRPC1/4/5 channels provide valuable hints to comprehend the functional importance of these ion channels in native cells and in vivo animal models. Importantly, human diseases and disorders mediated by these proteins can be studied using these compounds to perhaps initiate drug discovery efforts to develop novel therapeutic agents.

TRPC4/TRPC5 channels mediate adverse reaction to the cancer cell cytotoxic agent (-)-Englerin A.

(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg-1 i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation.

Tonantzitlolone is a nanomolar potency activator of transient receptor potential canonical 1/4/5 channels.

BACKGROUND AND PURPOSE: The diterpene ester tonantzitlolone (TZL) is a natural product, which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-englerin A, and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the 1, 4 and 5 type (TRPC1/4/5 channels). EXPERIMENTAL APPROACH: The effects of TZL on renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK293 cells overexpressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2, or CHO cells overexpressing TRPV4 were studied by determining changes in intracellular Ca2+ , or whole-cell or excised membrane patch-clamp electrophysiology. KEY RESULTS: TZL induced an elevation of intracellular Ca2+ in A498 cells, similar to that evoked by englerin A. TZL activated overexpressed channels with EC50 values of 123 nM (TRPC4), 83 nM (TRPC5), 140 nM (TRPC4-TRPC1) and 61 nM (TRPC5-TRPC1). These effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca2+ entry or overexpressed TRPC3, TRPV4 or TRPM2 channels in HEK 293 cells. CONCLUSIONS AND IMPLICATIONS: TZL is a novel potent agonist for TRPC1/4/5 channels, which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell.

Remarkable Progress with Small-Molecule Modulation of TRPC1/4/5 Channels: Implications for Understanding the Channels in Health and Disease.

Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na⁺, K⁺ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes.

Identification of an (-)-englerin A analogue, which antagonizes (-)-englerin A at TRPC1/4/5 channels.

BACKGROUND AND PURPOSE: (-)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma cells. It achieves its effects by activation of transient receptor potential canonical (TRPC)4/TRPC1 heteromeric channels. It is also an agonist at channels formed by the related protein, TRPC5. Here, we sought an EA analogue, which might enable a better understanding of these effects of EA. EXPERIMENTAL APPROACH: An EA analogue, A54, was synthesized by chemical elaboration of EA. The effects of EA and A54 on the activity of human TRPC4 or TRPC5 channels overexpressed on A498 and HEK 293 cells were investigated, firstly, by measuring intracellular Ca2+ and, secondly, current using whole-cell patch clamp recordings. KEY RESULTS: A54 had weak or no agonist activity at endogenous TRPC4/TRPC1 channels in A498 cells or TRPC4 or TRPC5 homomeric channels overexpressed in HEK 293 cells. A54 strongly inhibited EA-mediated activation of TRPC4/TRPC1 or TRPC5 and weakly inhibited activation of TRPC4. Studies of TRPC5 showed that A54 shifted the EA concentration-response curve to the right without changing its slope, consistent with competitive antagonism. In contrast, Gd3+ -activated TRPC5 or sphingosine-1-phosphate-activated TRPC4 channels were not inhibited but potentiated by A54. A54 did not activate TRPC3 channels or affect the activation of these channels by the agonist 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS AND IMPLICATIONS: This study has revealed a new tool compound for EA and TRPC1/4/5 channel research, which could be useful for characterizing endogenous TRPC1/4/5 channels and understanding EA-binding sites and their physiological relevance.

Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels.

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

(-)-Englerin A-evoked Cytotoxicity Is Mediated by Na+ Influx and Counteracted by Na+/K+-ATPase.

(-)-Englerin A ((-)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca2+-permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca2+ overload. Here we show that this is not the case and that the effect of (-)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (-)-EA cytotoxicity; however, although TRPC4 was necessary for the (-)-EA-evoked Ca2+ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca2+ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (-)-EA, and (-)-EA-evoked cell death was dependent upon entry of the monovalent cation Na+ We therefore hypothesized that Na+/K+-ATPase might act protectively by counteracting the Na+ load resulting from sustained Na+ entry. Indeed, inhibition of Na+/K+-ATPase by ouabain potently and strongly increased (-)-EA-evoked cytotoxicity. The data suggest that (-)-EA achieves cancer cell cytotoxicity by inducing sustained Na+ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (-)-EA can be potentiated by Na+/K+-ATPase inhibition.

The therapeutic agents that target ATP-sensitive potassium channels.

ATP-sensitive potassium (K(ATP)) channels are a major drug target for the treatment of type-2 diabetes. K(ATP) channels are ubiquitously expressed and link the metabolic state to electrical excitability. In pancreatic ß-cells, K(ATP) channels are crucial in the regulation of glucose-induced insulin secretion. Also, K(ATP) channels are involved in the protection against neuronal seizures and ischaemic stress in the heart, brain and in the regulation of vascular smooth muscle tone. Functional K(ATP) channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family, and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmaceutical agonists and antagonists, SUR allosterically regulates channel gating. The allosteric communication pathways between these two heterologus proteins in K(ATP) channels are still poorly understood. This review will highlight the therapeutic agents that target K(ATP) channels and are used to treat diabetes and cardiovascular diseases.

Natural and synthetic flavonoid modulation of TRPC5 channels.

BACKGROUND AND PURPOSE: The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. EXPERIMENTAL APPROACH: Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. KEY RESULTS: Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide-evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC50 0.45 µM). Galangin also inhibited lanthanide-evoked TRPC5-mediated current in whole-cell and outside-out patch recordings. In differentiated 3T3-L1 cells, it inhibited constitutive and lanthanide-evoked calcium entry through endogenous TRPC5-containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure-activity relationship studies with natural and synthetic flavonols, we designed 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12), which inhibited lanthanide-evoked TRPC5 activity with an IC50 of 0.28 µM. AM12 also inhibited TRPC5 activity evoked by the agonist (-)-Englerin A and was effective in excised outside-out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1-TRPC5 heteromeric channels was weaker. CONCLUSIONS AND IMPLICATIONS: The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.

Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.

In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge.

ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.

Relative contribution of different transmembrane segments to the CFTR chloride channel pore.

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.