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How many Orai's does it take to make a CRAC channel?

Thompson JL, Shuttleworth TJ - Sci Rep (2013)

Bottom Line: CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores.CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits.Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

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The apparent selectivity properties of the expressed Orai hexamer are influenced by the major external cation.(a) Mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM NMDG+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) Histogram comparing of the effects of changing external calcium concentrations from 10 mM to 0.1 mM on inward currents measured at −80 mV, in cells expressing either the concatenated Orai1 hexamer or the Orai1 tetramer.
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f2: The apparent selectivity properties of the expressed Orai hexamer are influenced by the major external cation.(a) Mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM NMDG+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) Histogram comparing of the effects of changing external calcium concentrations from 10 mM to 0.1 mM on inward currents measured at −80 mV, in cells expressing either the concatenated Orai1 hexamer or the Orai1 tetramer.

Mentions: To further examine the nature of the currents induced on expression of the hexameric Orai1 construct, we examined the effect of changing the major external cation from Na+ to NMDG+ ([NMDG+] = 140 mM, [Ca2+] = 10 mM). Store-operated currents recorded under these conditions displayed characteristics similar to those seen with the Na+-containing extracellular solution, namely an inwardly-rectifying current-voltage relationship with significant outward currents above the reversal potential of +20 mV. As noted above, this is indicative of the presence of a modest permeability to Cs+ ions (Fig. 2a). However, in marked contrast to the currents recorded in the Na+-based external solution, reducing extracellular Ca2+ from 10 mM to 0.1 mM now virtually eliminated this current (greater than 90% reduction at −80 mV) (Fig. 2a). These results indicate that the inward current seen with NMDG+ as the extracellular monovalent cation predominantly reflects the inward flux of Ca2+ ions. The fundamental differences in conductance properties of the channels formed by these various constructs are summarized in Fig. 2b which compares the effect of changing the external Ca2+ concentration from 10 mM to 0.1 mM, on the magnitude of inward store-operated currents measured at −80 mV in cells expressing either the concatenated hexamer or the concatenated tetramer.


How many Orai's does it take to make a CRAC channel?

Thompson JL, Shuttleworth TJ - Sci Rep (2013)

The apparent selectivity properties of the expressed Orai hexamer are influenced by the major external cation.(a) Mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM NMDG+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) Histogram comparing of the effects of changing external calcium concentrations from 10 mM to 0.1 mM on inward currents measured at −80 mV, in cells expressing either the concatenated Orai1 hexamer or the Orai1 tetramer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675454&req=5

f2: The apparent selectivity properties of the expressed Orai hexamer are influenced by the major external cation.(a) Mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM NMDG+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) Histogram comparing of the effects of changing external calcium concentrations from 10 mM to 0.1 mM on inward currents measured at −80 mV, in cells expressing either the concatenated Orai1 hexamer or the Orai1 tetramer.
Mentions: To further examine the nature of the currents induced on expression of the hexameric Orai1 construct, we examined the effect of changing the major external cation from Na+ to NMDG+ ([NMDG+] = 140 mM, [Ca2+] = 10 mM). Store-operated currents recorded under these conditions displayed characteristics similar to those seen with the Na+-containing extracellular solution, namely an inwardly-rectifying current-voltage relationship with significant outward currents above the reversal potential of +20 mV. As noted above, this is indicative of the presence of a modest permeability to Cs+ ions (Fig. 2a). However, in marked contrast to the currents recorded in the Na+-based external solution, reducing extracellular Ca2+ from 10 mM to 0.1 mM now virtually eliminated this current (greater than 90% reduction at −80 mV) (Fig. 2a). These results indicate that the inward current seen with NMDG+ as the extracellular monovalent cation predominantly reflects the inward flux of Ca2+ ions. The fundamental differences in conductance properties of the channels formed by these various constructs are summarized in Fig. 2b which compares the effect of changing the external Ca2+ concentration from 10 mM to 0.1 mM, on the magnitude of inward store-operated currents measured at −80 mV in cells expressing either the concatenated hexamer or the concatenated tetramer.

Bottom Line: CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores.CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits.Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

Show MeSH