A horde of cation-nonselective ion channel clans populate cell membranes. Their number is further augmented by removing extracellular calcium ([Ca2+]o), turning well-behaved Ca2+-selective channels into less discriminating sieves (Kostyuk and Krishtal, 1977; Almers and McCleskey, 1984). The Ca2+ ion stems the flood of ions moving through many cation channels by lodging in their throats, obstructing the path for monovalent ions by clinging to acidic groups in the narrow pore (Tsien et al., 1987; Hille, 2001). The trick of reducing [Ca2+]o uncovers channels whose conductances are inconveniently low in physiological solution. One of the most frustratingly low conductance Ca2+ channels has been the Ca2+ release–activated channel (CRAC) channel (Parekh and Penner, 1997; Lewis, 1999). The CRAC current is typically induced by the “store depletion” protocol in which thapsigargin, inositol trisphosphate (IP3), and/or dialysis using a Ca2+ chelator slowly evokes an anemic 10–50 pA of whole-cell inward current. Analysis of CRAC current variance estimates CRAC single channel conductance (even in 110 mM [Ca2+]o) of <30 fS, outside the reach of patch clamp measurements of reasonable bandwidth (Zweifach and Lewis, 1993).

Hoth and Penner (1993) and Lepple-Wienhues and Cahalan (1996) recorded a transient monovalent CRAC current upon external divalent removal. Subsequently, Kerschbaum and Cahalan (1998) recorded a much larger and noninactivating monovalent current when internal Mg2+ was also omitted. Thus, it was logical to look for the highly Ca2+-selective CRAC channel single channel conductance by lowering [Ca2+]o. Cahalan and colleagues reasonably assumed they had uncovered the CRAC single channel conductance by removing divalents from both sides of the CRAC-loaded Jurkat T lymphocyte in whole-cell recordings (Kerschbaum and Cahalan, 1999; see also Lepple-Wienhues and Cahalan, 1996; Kerschbaum and Cahalan, 1998; Fomina et al., 2000). The presumed CRAC single channels in monovalent solution were 35–40 pS in size, leading to estimates of only 15–150 channels in resting and activated T cells, respectively (Fomina et al., 2000). Identification of the presumed ∼40 pS CRAC monovalent single channel conductance was a hopeful development as a useful new tool for identification of CRAC channels. The monovalent whole-cell conductance was 40 times higher than in [Ca2+]o-containing solutions, blocked in a voltage-dependent manner by 10 μM [Ca2+]o, and was inactivated by intracellular Mg2+ ions. But inactivation of the presumed monovalent CRAC current by [Mg2+]i, turns out to be very important, and ultimately distinguishes ICRAC from the newly identified monovalent current they had unwittingly revealed.

Now, three recently published papers clearly show that the monovalent current, proposed by Cahalan and colleagues to be ICRAC, differs significantly from bona fide ICRAC (Hermosura et al., 2002; Prakriya and Lewis, 2002; Kozak and Cahalan, 2002, this issue). Hermosura et al. (2002) showed that the monovalent current (which they called MagNum, for magnesium- and nucleotide-regulated metal current) could be separated from ICRAC by its kinetics and internal Mg2+ sensitivity. In a very detailed study in the May issue of the Journal of General Physiology, Prakriya and Lewis (2002) clearly separate CRAC and from the Mg2+-inhibited current (which they called MIC). Finally, and again in the Journal of General Physiology, Kozak and Cahalan (2002) also show that the monovalent current is composed primarily of MIC channels that have intrinsic selectivity properties and can be selectively blocked or induced to run down.

It is apparent from the careful work of Prakriya and Lewis (2002) that ICRAC's single channel conductance, even in monovalent conditions, is <1 pS. This is an important finding for several reasons. First, Yue et al. (2001) proposed that the Ca2+-selective TRPV6 is part of the CRAC channel, based partly on the similar monovalent single channel conductance measured in Cahalan and colleagues' previous work (e.g., Kerschbaum and Cahalan, 1999) and on TRPV6's unique inwardly rectifying (CRAC-like) whole-cell current-voltage relation. The small conductance of CRAC, even under monovalent conditions, mitigates against the conclusion that TRPV6 (CaT1) encodes the CRAC pore itself. It does not exclude the possibility that TRPV6 is part of a heteromeric protein comprising CRAC (but see also Voets et al., 2001). Second, the low conductance in monovalent conditions lacking Ca2+ brings up an interesting point about the highly Ca2+-selective CRAC channel. Most Ca2+channels (in particular the large class of voltage-dependent CaVs) are selective for Ca2+ because they have one or more high affinity sites for Ca2+ in the mouth of the pore, usually implemented by a ring of negatively charged (glutamates or aspartate) amino acids (Ellinor et al., 1995). CRAC somehow accomplishes its selectivity for Ca2+ without the need to bind external Ca2+ ions in its presumed pore. It is tantalizing to speculate that this feature implies a unique channel or transporter-like structure for CRAC. But further speculation about how this is accomplished must await unequivocal identification of the molecule encoding CRAC.

If MIC is not CRAC, what is it? Jaded electrophysiologists are usually more annoyed than pleased by the appearance of small inward whole-cell currents, because they are difficult to distinguish from artifactual leak currents. With the wealth of K+ channels that can produce large currents at positive membrane potentials, they are also inured to the gradual appearance of yet another outward current. But it is this combination of features that typifies many of the transient receptor potential TRP channels (Fig. 1 A). When expressed in heterologous systems, many of the known TRP channels produce relatively nonselective currents, small at potentials <0 mV, but steeply rectifying and large at positive potentials (Clapham et al., 2001). This current-voltage relationship is also grossly similar to that recorded from TRPV1–4, TRPM7, and TRPM8-expressing cells (Fig. 1, A and B). The short answer to the question posed at the beginning of this paragraph is that MIC is most likely encoded by TRPM7 (TRP-PLIK, ChaK1, LTRPC7) or its close relative TRPM6 (ChaK2). The argument that MIC is encoded by TRPM7 is best made by comparing the detailed work of the three original MIC papers and three papers on TRPM7 (Runnels et al., 2001, 2002; Nadler et al., 2001), but the relevant features that separate MIC from CRAC and align it with TRPM7 are summarized in Table I.

The first member of the TRPM group to be functionally expressed as an ion channel, TRPM7, has the unique feature of also being itself a protein kinase. TRPM7 is an 1,863 amino acid–containing protein identified in a yeast two-hybrid screen as a protein interacting with PLC-β1 (Runnels et al., 2001, 2002). TRPM7 is a relatively nonselective, ∼80-pS (chord conductance at 100 mV) channel that exhibits a steeply outwardly rectifying conductance when expressed in mammalian cells (Nadler et al. 2001; Runnels et al., 2001). The mechanism of its activation is still unclear, but does not require the kinase domain for activity (Runnels et al., 2002; unpublished data). Receptor-mediated activation of PLC by hormones or growth factors inhibits channel activity by hydrolyzing and reducing local PIP2 concentrations (Runnels et al., 2002). Most important for this story, TRPM7 is inhibited by [Mg2+]i (IC50 ∼0.5 mM; Nadler et al., 2001) and appears to be expressed in every cell examined, including blood cells and cell lines commonly used for expression. Like MIC, TRPM7's Mg2+ inhibition is not a simple blockade and may involve another diffusible molecule.

In hindsight, ICRAC and IMIC are not very similar. But, the apparently wide distribution of TRPM7 (presumably encoding IMIC) and its presence in cell lines used for expressing novel channel genes, will continue to entrap unwary investigators. Given the varied state of MIC activation at the initiation of whole-cell recordings and the small size of inward currents, leak subtraction is particularly hazardous. Until the discovery of toxins or other agents that specifically block IMIC, one can eliminate MIC by including high [Mg2+]i in the pipette (e.g., 10 mM). However, this alone will not solve the problem of separating MIC from other ion channels that are potentially Mg2+-sensitive, and confounds the use of Mg2+ binding moieties such as ATP (and thus the regulation of PIP2). But the most difficult issue for future recordings will be separating MIC from other TRPs, particularly TRPV1–4 and TRPM8.

What can we learn from these MIC, CRAC, and TRPM7 studies? Almost all molecularly identified TRPs, as well as CRAC, dribble Ca2+ into cells at potentials <0 mV. They are activated by as yet unclear mechanisms, many involving the complex phospholipase C and phosphatidylinositol pathways. Evidence is steadily emerging that these channels, eerily reminiscent of glutamategic channels, are localized in signal transduction complexes by scaffolding proteins. My guess is that CRAC and TRP channels are tethered sources that provide localized Ca2+ increases for spatially defined signal transduction pathways. It seems doubtful that their main purpose is the repletion of intracellular Ca2+ stores. But for CRAC addicts the question remains, what gene or genes encode CRAC?

Almers, W., and E.W. McCleskey.
. Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore.
J. Physiol.
Clapham, D.E., L.W. Runnels, and C. Strübing.
. The TRP ion channel family.
Nat. Rev. Neurosci.
Ellinor, P.T., J. Yang, W.A. Sather, J.F. Zhang, and R.W. Tsien.
. Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions.
Fomina, A.F., C.M. Fanger, J.A. Kozak, and M.D. Cahalan.
. Single channel properties and regulated expression of Ca2+ release-activated Ca2+ (CRAC) channels in human T cells.
J. Cell Biol.
Hermosura, M.C., M.K. Monteilh-Zoller, A.M. Scharenberg, R. Penner, and A. Fleig.
. Dissociation of the store-operated calcium current ICRAC and the Mg2+-nucleotide-regulated metal ion current MagNuM.
J. Physiol.
Hille, B. 2001. Ionic channels of excitable membranes. Sinauer Associates, Inc., Sunderland, MA.
Hoth, M., and R. Penner.
. Calcium release-activated calcium current in rat mast cells.
J. Physiol.
Kerschbaum, H.H., and M.D. Cahalan.
. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes.
J. Gen. Physiol.
Kerschbaum, H.H., and M.D. Cahalan.
. Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes.
Kostyuk, P.G., and O.A. Krishtal.
. Effects of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones.
J. Physiol
Kozak, J.A., and M.D. Cahalan.
. Separation and distinct properties of divalent and monovalent currents through CRAC and BC channels in RBL mast cells.
J. Gen. Physiol.
Lepple-Wienhues, A., and M.D. Cahalan.
. Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.
Biophys. J.
Lewis, R.S.
. Store-operated calcium channels.
Adv. Sec. Mess. Phosphoprot. Res.
Nadler, M.J., M.C. Hermosura, K. Inabe, A.L. Perraud, Q. Zhu, A.J. Stokes, T. Kurosaki, J.P. Kinet, R. Penner, A.M. Scharenberg, and A. Fleig.
. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.
Parekh, A.B., and R. Penner.
. Store depletion and calcium influx.
Physiol. Rev.
Prakriya, M., and R. Lewis.
. Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.
J. Gen. Physiol.
Runnels, L.W., L. Yue, and D.E. Clapham.
. TRP-PLIK, a bifunctional protein with kinase and ion channel activities.
Runnels, L.W., L. Yue, and D.E. Clapham.
. The TRPM7 channel is inactivated by PIP2 hydrolysis.
Nat. Cell Biol.
Tsien, R.W., P. Hess, E.W. McCleskey, and R.L. Rosenberg.
. Calcium channels: mechanisms of selectivity, permeation, and block.
Annu. Rev. Biophys. Chem.
Voets, T., J. Prenen, A. Fleig, R. Vennekens, H. Watanabe, G.J. Hoenderop, R.J. Bindels, G. Droogmans, R. Penner, B. Nilius.
. CaT1 and the calcium-release activated calcium channel manifest distinct pore properties.
J. Biol. Chem.
Yue, L., J.B. Peng, M.A. Hediger, and D.E. Clapham.
. CaT1 manifests the pore properties of the calcium-release-activated calcium channel.
Zweifach, A., and R.S. Lewis.
. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores.
Proc. Natl. Acad. Sci. USA.