Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes

doi:10.1016/S0143416002001914

Cell Calcium

Volume 32, Issues 5–6, November–December 2002, Pages 379-391

https://doi.org/10.1016/S0143416002001914 Get rights and content

Abstract

CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward.

Like all Ca²⁺-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na⁺ currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I_CRAC in divalent-free external solution, notably omission of Mg²⁺/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg²⁺-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.

Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is ∼200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.

Section snippets

INTRODUCTION

Store-operated (capacitative) calcium influx, in which a fall in endoplasmic reticulum (ER) calcium content opens calcium channels in the plasma membrane, is one of the more widespread mechanisms whereby mammalian non-excitable cells can increase their intracellular free calcium concentration 1., 2.. Calcium entry through store-operated calcium channels is important for regulating a wide spectrum of kinetically distinct processes ranging from exocytosis and enzymatic activity to gene

BASIC FEATURES OF CRAC CHANNELS

With Ca²⁺ as the charge carrier, I_CRAC is a non-voltage-gated, inwardly rectifying current with a very positive reversal potential (>+60 mV), the latter indicating that the current is very selective for Ca²⁺ 1., 7.. Although Na⁺ ions outnumber Ca²⁺ by 70:1, neither the extent of I_CRAC nor the positive reversal potential appear altered by substituting external Na⁺ for organic cations like NMDG⁺ in the presence of physiological levels of external Ca²⁺ (1–2 mM) [10]. Moreover, removal of external Ca

MONOVALENT PERMEATION THROUGH CRAC CHANNELS

Fig. 1 compares the whole-cell current in an RBL-1 cell dialysed with InsP₃+10 mM EGTA and bathed in either external solution containing 10 mM Ca²⁺ or in divalent-free external solution with Na⁺ as the major carrier of inward current. Although the amplitude of the current is ∼sevenfold larger in divalent-free solution than in the presence of Ca²⁺, both develop with similar time-courses (Fig. 1A). The I–V curves, taken at steady-state, are shown in Fig. 1B. Inward rectification is seen in both

ANOMALOUS MOLE FRACTION

Ca²⁺-selective channels show concentration-dependent permeability ratios, which are indicative of multi-ion pores [29]. Such anomalous mole fraction behaviour is seen in both divalent–divalent and monovalent–divalent mixtures. The conductance of CRAC channels is higher in external Ca²⁺ than equimolar Ba²⁺ solutions [22], but with mixtures of the two ions conductance falls to a level less than that seen in pure Ca²⁺ and Ba²⁺ solutions. Similarly, increasing external calcium to the micromolar

SINGLE CRAC CHANNEL CONDUCTANCE

With Ca²⁺ ions as the charge carrier, the single CRAC channel conductance is very low. Over a wide variety of voltages, Hoth and Penner did not detect any increase in macroscopic variance as I_CRAC developed in mast cells [12]. They estimated a single-channel conductance substantially lower than 1 pS. Using stationary noise analysis in isotonic Ca²⁺, Zweifach and Lewis estimated a unitary chord conductance of 24 fS in Jurkat T lymphocytes [21]. This is almost three orders of magnitude smaller than

MagNuM

The discovery of a Mg²⁺/Mg-ATP-regulated current in several different non-excitable cells has raised the possibility that whole-cell currents previously attributed to I_CRAC might be contaminated by biophysically distinct Mg-ATP-regulated channels under certain experimental conditions. We begin with a brief description of the relevant features of this Mg²⁺/Mg-ATP-regulated current.

Overexpression of recombinant TRP-PLIK/LTRPC7 (now called TRPM7), a member of the LTRPC family of ion channels,

TRPV6

Despite intense investigation, the gene(s) encoding the CRAC channel remains elusive. Various members of the TRP family of proteins have been proposed to account for I_CRAC, but the currents arising from the expression of these proteins in recombinant systems differ from native I_CRAC 1., 6.. TRPV6 (ECaC2/CaT1) and the highly homologous TRPV5 (ECaC1/CaT2) are both Ca²⁺-selective channels which exhibit strong inward rectification, a positive reversal potential for Ca²⁺, a similar divalent cation

Acknowledgements

We thank Reinhold Penner and Bernd Nilius for sending us the I–V relationship for TRPV6 and Reinhold Penner for the I–V of MagNuM, both used in Table 1.

References (56)

M Partiseti et al.
The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency
J. Biol. Chem.
(1994)
C Zitt et al.
The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry
Prog. Neurobiol.
(2002)
F.J Braun et al.
Stable activation of single Ca²⁺ release-activated Ca²⁺ channels in divalent cation-free solutions
J. Biol. Chem.
(2001)
T Voets et al.
CaT1 and the calcium release-activated calcium channel manifest distinct pore properties
J. Biol. Chem.
(2001)
A Lepple-Wienhues et al.
Conductance and permeation of monovalent cations through depletion-activated Ca²⁺ channels (I_CRAC) in Jurkat T cells
Biophys. J.
(1996)
B Nilius et al.
The single pore residue Asp542 determines Ca²⁺ permeation and Mg²⁺ block of the epithelial Ca²⁺ channel
J. Biol. Chem.
(2001)
R Vennekens et al.
Permeation and gating properties of the novel epithelial Ca²⁺ channel
J. Biol. Chem.
(2000)
J.G Hoenderop et al.
Molecular identification of the apical Ca²⁺ channel in 1,25-dihydroxyvitamin D₃-responsive epithelia
J. Biol. Chem.
(1999)
J.B Peng et al.
Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption
J. Biol. Chem.
(1999)
O.H Petersen et al.
Calcium signalling: store-operated channel found at last
Curr. Biol.
(2001)

Cited by (42)

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury
2022, Journal of Biological Chemistry
Citation Excerpt :
To examine if the Orai1 channel is functional in podocytes, we carried out electrophysiology experiments and measured the channel currents in response to the channel activators and blockers. Because SOC is more conductive to monovalent cations over divalent cations (26–29), we measured Na+ currents using the mode of the whole-cell patch. As shown in Figure 1C, thapsigargin (TG), a well-known activator of SOC induced robust inward currents, which were greatly reversed by La3+ (2 μM), an inhibitor of SOC (25, 30).
Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.
Nitric oxide displays a biphasic effect on calcium dynamics in microglia
2021, Nitric Oxide - Biology and Chemistry
Citation Excerpt :
To further examine the impact of NO on SOCCs we utilized TG to evoke SOCE. We demonstrated that the SOCC inhibitors 2APB or SKF96365 [5,8,24,32,43,45] could significantly abolish the TG-mediated SOCE in murine microglia. Therefore, we further utilized TG to directly examine the effects of NO on SOCE in BV2 microglia.
Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS^−/−) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS^−/− microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia.
Authentic CRAC channel activity requires STIM1 and the conserved portion of the Orai N terminus
2018, Journal of Biological Chemistry
Citation Excerpt :
Orai channels conduct small monovalent ions, such as Na+, Li+, or K+, as long as the monovalent solution lacks divalent ions. Monovalent Orai currents are inhibited by Ca2+ concentrations in the micromolar range (43–48). Upon the switch from a Ca2+-containing to a divalent-free (DVF) Na+-containing solution, CRAC/Orai currents have been reported to increase 5-fold or even more (42, 46, 49–51), displaying another prominent CRAC channel hallmark.
Calcium (Ca²⁺) is an essential second messenger required for diverse signaling processes in immune cells. Ca²⁺ release-activated Ca²⁺ (CRAC) channels represent one main Ca²⁺ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca²⁺ sensor in the endoplasmic reticulum, and the Ca²⁺ ion channel Orai in the plasma membrane. After Ca²⁺ store depletion, STIM1 and Orai couple to each other, allowing Ca²⁺ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca²⁺ selectivity, an increase in current density when switching from a Ca²⁺-containing solution to a divalent-free Na⁺ one, and fast Ca²⁺-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.
Mitofusin 2 regulates STIM1 migration from the Ca<sup>2+</sup> store to the plasma membrane in cells with depolarized mitochondria
2011, Journal of Biological Chemistry
Citation Excerpt :
Therefore, mitochondrial depolarization impairs STIM1 trafficking even in the absence of a cytoplasmic Ca2+ rise and therefore in the absence of the ability of the organelle to take up Ca2+. Finally, we overexpressed Orai1 and STIM1 and then measured the Na+ flux through the CRAC channels that occurs when cells are exposed to divalent-free external solution (51, 54–56). Following dialysis with InsP3 and 10 mm BAPTA, a large Na+ current developed (Fig. 3D), which showed the characteristics of Na+ flux through CRAC channels (Fig. 3E; inwardly rectifying current-voltage relationship, reversal potential of approximately +60 mV, low current noise).
Store-operated Ca²⁺ channels in the plasma membrane (PM) are activated by the depletion of Ca²⁺ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca²⁺ influx pathway. After store emptying, the ER Ca²⁺ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca²⁺ release-activated Ca²⁺ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca²⁺ release-activated Ca²⁺ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca²⁺ entry, thereby reducing cellular Ca²⁺ overload.
A novel native store-operated calcium channel encoded by Orai3: Selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells
2010, Journal of Biological Chemistry
Store-operated calcium (Ca²⁺) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca²⁺ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca²⁺ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ current (I_CRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca²⁺ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I_CRAC in estrogen receptor-positive (ER⁺) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER⁻) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER⁺ breast cancer cells represent the first example where the native SOCE pathway and I_CRAC are mediated by Orai3. Future studies implicating Orai3 in ER⁺ breast cancer progression might establish Orai3 as a selective target in therapy of ER⁺ breast tumors.
Native store-operated Ca<sup>2+</sup> influx requires the channel function of Orai 1 and TRPC1
2009, Journal of Biological Chemistry
Citation Excerpt :
This resulted in a small current with shallow inward rectification and a reversal potential of about 10 mV. This is different from the properties of CRAC channels in T and RBL cells (33). The analysis in Fig. 1 suggests that in the minority of our HEK cells, the native SOCs current is carried mostly by Orai1 and that it is modulated or enhanced by TRPC1.
With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca²⁺ influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.

View all citing articles on Scopus

View full text

Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes

Abstract

Section snippets

INTRODUCTION

BASIC FEATURES OF CRAC CHANNELS

MONOVALENT PERMEATION THROUGH CRAC CHANNELS

ANOMALOUS MOLE FRACTION

SINGLE CRAC CHANNEL CONDUCTANCE

MagNuM

TRPV6

Acknowledgements

J. Biol. Chem.

Prog. Neurobiol.

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Biol.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Store depletion and calcium influx

Physiol. Rev.

Mechanisms of capacitative calcium entry

J. Cell Sci.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

Proc. Natl. Acad. Sci. U.S.A.

Calcium signaling and acute pancreatitis: specific response to a promiscuous messenger

Proc. Natl. Acad. Sci. U.S.A.

Calcium signaling mechanisms in T lymphocytes

Annu. Rev. Immunol.

Depletion of intracellular calcium stores activates a calcium current in mast cells

Nature

Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells

Mol. Cell. Biol.

Substantial depletion of the intracellular Ca2+ stores is required for macroscopic activation of the Ca2+ release-activated Ca2+ current in rat basophilic leukaemia cells

J. Physiol.

Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca2+ current ICRAC

EMBO J.

Calcium release-activated calcium current in rat mast cells

J. Physiol.

Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes

Science

Substantial depletion of the intracellular Ca²⁺ stores is required for macroscopic activation of the Ca²⁺ release-activated Ca²⁺ current in rat basophilic leukaemia cells

Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca²⁺ current I_CRAC

Single-channel recording of a store-operated Ca²⁺ channel in Jurkat T lymphocytes