Limits...
Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.

Prakriya M, Lewis RS - J. Gen. Physiol. (2002)

Bottom Line: Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity.These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes.Store depletion does not activate MIC channels, nor does store refilling deactivate them.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.

Show MeSH

Related in: MedlinePlus

ICRAC and the large monovalent current exhibit different sensitivities to SKF 96365. All cells were pretreated with 1 μM TG. In B and C, the recordings began after the monovalent current had activated to a steady-state level, as described in Fig. 2. Both currents were measured during steps to −110 mV. (A) Inhibition of ICRAC by SKF 96365 (20 μM). Inhibition and recovery followed exponential time courses with time constants of 17.5 and 19 s, respectively. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. External: 20 mM Ca2+. (B) The large monovalent current is relatively insensitive to 20 μM SKF 96365. Internal solution: MGF. (C) Even after nearly complete inhibition of ICRAC by SKF 96365 (20 μM), removal of external divalent ions causes the monovalent current to rise rapidly to control levels. Thus, the insensitivity of the monovalent current to SKF 96365 is not explained by a failure to block CRAC channels under DVF conditions. Internal solution: MGF.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233817&req=5

fig4: ICRAC and the large monovalent current exhibit different sensitivities to SKF 96365. All cells were pretreated with 1 μM TG. In B and C, the recordings began after the monovalent current had activated to a steady-state level, as described in Fig. 2. Both currents were measured during steps to −110 mV. (A) Inhibition of ICRAC by SKF 96365 (20 μM). Inhibition and recovery followed exponential time courses with time constants of 17.5 and 19 s, respectively. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. External: 20 mM Ca2+. (B) The large monovalent current is relatively insensitive to 20 μM SKF 96365. Internal solution: MGF. (C) Even after nearly complete inhibition of ICRAC by SKF 96365 (20 μM), removal of external divalent ions causes the monovalent current to rise rapidly to control levels. Thus, the insensitivity of the monovalent current to SKF 96365 is not explained by a failure to block CRAC channels under DVF conditions. Internal solution: MGF.

Mentions: To further establish differences between the large monovalent current and CRAC channels and find conditions that could be used to isolate each current, we compared their sensitivities to SKF 96365 and 2-APB. SKF 96365 is an imidazole antimycotic compound that inhibits CRAC channels and several other SOCs with IC50 values of 0.6–16 μM (Franzius et al., 1994; Christian et al., 1996a). Application of 20 μM SKF 96365 caused robust and partially reversible inhibition of ICRAC (Fig. 4 A). On average, the peak current amplitude was diminished by 87 ± 4% with a time constant of 17 ± 7 s (n = 6). In contrast, the same concentration of SKF 96365 had very little effect on the large monovalent current (Fig. 4 B), inhibiting by only 10 ± 2% after 120 s of exposure (n = 4). Two results argue that the resistance of the monovalent current to block by SKF 96365 is not due to DVF conditions per se. First, with 20 mM Ca2+ present, the compound also failed to inhibit the outwardly rectifying current seen in Fig. 2 D, which we believe is mediated by the same channels that conduct the large monovalent current under DVF conditions (see below). Furthermore, complete inhibition of ICRAC by drug application in the presence of Ca2+ did not affect or delay the appearance of the large monovalent current upon removal of extracellular divalents (Fig. 4 C). Thus, CRAC channels and the large monovalent current are distinctly different in their sensitivities to inhibition by SKF 96365.


Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.

Prakriya M, Lewis RS - J. Gen. Physiol. (2002)

ICRAC and the large monovalent current exhibit different sensitivities to SKF 96365. All cells were pretreated with 1 μM TG. In B and C, the recordings began after the monovalent current had activated to a steady-state level, as described in Fig. 2. Both currents were measured during steps to −110 mV. (A) Inhibition of ICRAC by SKF 96365 (20 μM). Inhibition and recovery followed exponential time courses with time constants of 17.5 and 19 s, respectively. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. External: 20 mM Ca2+. (B) The large monovalent current is relatively insensitive to 20 μM SKF 96365. Internal solution: MGF. (C) Even after nearly complete inhibition of ICRAC by SKF 96365 (20 μM), removal of external divalent ions causes the monovalent current to rise rapidly to control levels. Thus, the insensitivity of the monovalent current to SKF 96365 is not explained by a failure to block CRAC channels under DVF conditions. Internal solution: MGF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233817&req=5

fig4: ICRAC and the large monovalent current exhibit different sensitivities to SKF 96365. All cells were pretreated with 1 μM TG. In B and C, the recordings began after the monovalent current had activated to a steady-state level, as described in Fig. 2. Both currents were measured during steps to −110 mV. (A) Inhibition of ICRAC by SKF 96365 (20 μM). Inhibition and recovery followed exponential time courses with time constants of 17.5 and 19 s, respectively. Internal solution: Cs methanesulfonate/10 BAPTA/8 Mg2+. External: 20 mM Ca2+. (B) The large monovalent current is relatively insensitive to 20 μM SKF 96365. Internal solution: MGF. (C) Even after nearly complete inhibition of ICRAC by SKF 96365 (20 μM), removal of external divalent ions causes the monovalent current to rise rapidly to control levels. Thus, the insensitivity of the monovalent current to SKF 96365 is not explained by a failure to block CRAC channels under DVF conditions. Internal solution: MGF.
Mentions: To further establish differences between the large monovalent current and CRAC channels and find conditions that could be used to isolate each current, we compared their sensitivities to SKF 96365 and 2-APB. SKF 96365 is an imidazole antimycotic compound that inhibits CRAC channels and several other SOCs with IC50 values of 0.6–16 μM (Franzius et al., 1994; Christian et al., 1996a). Application of 20 μM SKF 96365 caused robust and partially reversible inhibition of ICRAC (Fig. 4 A). On average, the peak current amplitude was diminished by 87 ± 4% with a time constant of 17 ± 7 s (n = 6). In contrast, the same concentration of SKF 96365 had very little effect on the large monovalent current (Fig. 4 B), inhibiting by only 10 ± 2% after 120 s of exposure (n = 4). Two results argue that the resistance of the monovalent current to block by SKF 96365 is not due to DVF conditions per se. First, with 20 mM Ca2+ present, the compound also failed to inhibit the outwardly rectifying current seen in Fig. 2 D, which we believe is mediated by the same channels that conduct the large monovalent current under DVF conditions (see below). Furthermore, complete inhibition of ICRAC by drug application in the presence of Ca2+ did not affect or delay the appearance of the large monovalent current upon removal of extracellular divalents (Fig. 4 C). Thus, CRAC channels and the large monovalent current are distinctly different in their sensitivities to inhibition by SKF 96365.

Bottom Line: Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity.These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes.Store depletion does not activate MIC channels, nor does store refilling deactivate them.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.

Show MeSH
Related in: MedlinePlus