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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.

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Pharmacological evidence for monovalent currents through CRAC channels. ICRAC was activated by treatment with 1 μM TG for 5 min before seal formation (in A and B) or by passive depletion with 10 mM intracellular BAPTA (in C), with 10 mM intracellular Mg2+ to inhibit IMIC. (A) SKF 96365 (20 μM) inhibits both ICRAC and the transient monovalent current (under DVF conditions). Both currents recover following washout of the drug. (B) A low concentration of 2-APB (5 μM) enhances both ICRAC and the transient monovalent current by severalfold. (C) A high concentration of 2-APB (40 μM) significantly reduces both ICRAC and the transient monovalent current. The inhibition of both currents persists after washout of the drug.
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fig9: Pharmacological evidence for monovalent currents through CRAC channels. ICRAC was activated by treatment with 1 μM TG for 5 min before seal formation (in A and B) or by passive depletion with 10 mM intracellular BAPTA (in C), with 10 mM intracellular Mg2+ to inhibit IMIC. (A) SKF 96365 (20 μM) inhibits both ICRAC and the transient monovalent current (under DVF conditions). Both currents recover following washout of the drug. (B) A low concentration of 2-APB (5 μM) enhances both ICRAC and the transient monovalent current by severalfold. (C) A high concentration of 2-APB (40 μM) significantly reduces both ICRAC and the transient monovalent current. The inhibition of both currents persists after washout of the drug.

Mentions: The pharmacological profile of the transient Na+ current also closely matched that of ICRAC. As shown in Fig. 9 A, 20 μM SKF 96365 inhibited both currents to similar extents. On average, ICRAC was reduced by 84 ± 3% (n = 4), whereas the inactivating Na+ current was reduced by 74 ± 3% in the same cells. Moreover, 5 μM 2-APB potentiated both currents, enhancing ICRAC by 241 ± 41% (n = 7) and the peak amplitude of the transient Na+ current in the same cells by 185 ± 31% (Fig. 9 B). 2-APB also enhanced the steady-state component of the Na+ current by a similar amount (224 ± 26%; n = 6). These results suggest that the residual current remaining after the Na+ current has declined is also due to CRAC channels, a conclusion that is consistent with the observation that the peak and the residual currents reverse at the same potential (see below).


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

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

Pharmacological evidence for monovalent currents through CRAC channels. ICRAC was activated by treatment with 1 μM TG for 5 min before seal formation (in A and B) or by passive depletion with 10 mM intracellular BAPTA (in C), with 10 mM intracellular Mg2+ to inhibit IMIC. (A) SKF 96365 (20 μM) inhibits both ICRAC and the transient monovalent current (under DVF conditions). Both currents recover following washout of the drug. (B) A low concentration of 2-APB (5 μM) enhances both ICRAC and the transient monovalent current by severalfold. (C) A high concentration of 2-APB (40 μM) significantly reduces both ICRAC and the transient monovalent current. The inhibition of both currents persists after washout of the drug.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Pharmacological evidence for monovalent currents through CRAC channels. ICRAC was activated by treatment with 1 μM TG for 5 min before seal formation (in A and B) or by passive depletion with 10 mM intracellular BAPTA (in C), with 10 mM intracellular Mg2+ to inhibit IMIC. (A) SKF 96365 (20 μM) inhibits both ICRAC and the transient monovalent current (under DVF conditions). Both currents recover following washout of the drug. (B) A low concentration of 2-APB (5 μM) enhances both ICRAC and the transient monovalent current by severalfold. (C) A high concentration of 2-APB (40 μM) significantly reduces both ICRAC and the transient monovalent current. The inhibition of both currents persists after washout of the drug.
Mentions: The pharmacological profile of the transient Na+ current also closely matched that of ICRAC. As shown in Fig. 9 A, 20 μM SKF 96365 inhibited both currents to similar extents. On average, ICRAC was reduced by 84 ± 3% (n = 4), whereas the inactivating Na+ current was reduced by 74 ± 3% in the same cells. Moreover, 5 μM 2-APB potentiated both currents, enhancing ICRAC by 241 ± 41% (n = 7) and the peak amplitude of the transient Na+ current in the same cells by 185 ± 31% (Fig. 9 B). 2-APB also enhanced the steady-state component of the Na+ current by a similar amount (224 ± 26%; n = 6). These results suggest that the residual current remaining after the Na+ current has declined is also due to CRAC channels, a conclusion that is consistent with the observation that the peak and the residual currents reverse at the same potential (see below).

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