Store-operated Ca 2+ entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca 2+ sensors with calcium release–activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) 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.

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 2+ and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs + or Na + 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 2+ as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K + -based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca 2+ , both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca 2+ , a minimum of 30 nM internal Ca 2+ is required to cause partial TRPM2 activation with ADPR. However, 200 μM external Ca 2+ is as efficient as 1 mM Ca 2+ in TRPM2 activation, indicating an external Ca 2+ binding site important for proper channel function. Ca 2+ facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca 2+ . Furthermore, TRPM2 currents inactivate if intracellular Ca 2+ levels fall below 100 nM irrespective of extracellular Ca 2+ . The facilitatory effect of intracellular Ca 2+ is not mimicked by Mg 2+ , Ba 2+ , or Zn 2+ . Only Sr 2+ facilitates TRPM2 as effectively as Ca 2+ , but this is due to Sr 2+ -induced Ca 2+ release from internal stores rather than a direct effect of Sr 2+ itself. Together, these data demonstrate that cytosolic Ca 2+ regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 μM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca 2+ ] i and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca 2+ facilitates activation via calmodulin.

TRPM7 is a Ca 2+ - and Mg 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 2+ , the functional roles of intracellular levels of Mg·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·ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg 2+ . Mg·ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg 2+ and Ca 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 ≥ GTP ≥ UTP > ITP ≈ free Mg 2+ alone. These nucleotides also enhanced TRPM7 inhibition by free Mg 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 2+ -dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg·NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg 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 2+ binding site extrinsic to that domain.

Trace metal ions such as Zn 2+ , Fe 2+ , Cu 2+ , Mn 2+ , and Co 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 2+ - and Mg 2+ -permeable cation channel, whose activity is regulated by intracellular Mg 2+ and Mg 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 2+ and Ni 2+ , which both permeate TRPM7 up to four times better than Ca 2+ . Similarly, native MagNuM currents are also able to support Zn 2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn 2+ and Co 2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd 2+ , Ba 2+ , and Sr 2+ . Equimolar replacement studies substituting 10 mM Ca 2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn 2+ ≈ Ni 2+ >> Ba 2+ > Co 2+ > Mg 2+ ≥ Mn 2+ ≥ Sr 2+ ≥ Cd 2+ ≥ Ca 2+ , while trivalent ions such as La 3+ and Gd 3+ are not measurably permeable. With the exception of Mg 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 2+ , Co 2+ , or Ni 2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca 2+ and Mg 2+ , suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca 2+ release channel (in the membrane of internal Ca 2+ stores) and the dihydropyridine receptor (DHPR)-Ca 2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca 2+ channels, we expressed α 2 , β, and γ DHPR subunits and a chimeric DHPR α 1 subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca 2+ transient (CaT) in the absence of extracellular Ca 2+ ([Ca 2+ ] o ). However, in the presence of [Ca 2+ ] o , depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca 2+ ] i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca 2+ influx through Ca 2+ channels, whereas the second component resulted from Ca 2+ release through the RyR expressed in the membrane of internal Ca 2+ stores. However, the onset and the rate of Ca 2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca 2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca 2+ -induced Ca 2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.