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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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Selectivity properties of expressed hexameric and tetrameric Orai1 concatamers.(a) Mean (±SE, n = 6) I/V curves for store-operated currents recorded between −100 mV and +80 mV in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM Na+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) The corresponding mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 tetramer. (c) Western blot showing the expressed FLAG-tagged concatamers running at their appropriate expected molecular masses (approximately 195 kDa for the hexamer, and 130 kDa for the tetramer).
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f1: Selectivity properties of expressed hexameric and tetrameric Orai1 concatamers.(a) Mean (±SE, n = 6) I/V curves for store-operated currents recorded between −100 mV and +80 mV in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM Na+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) The corresponding mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 tetramer. (c) Western blot showing the expressed FLAG-tagged concatamers running at their appropriate expected molecular masses (approximately 195 kDa for the hexamer, and 130 kDa for the tetramer).

Mentions: Given the above contradictory findings, we chose to directly examine the functional properties of hexameric Orai1 channels in more detail. For this we generated a construct consisting of a concatenated hexameric assembly of full-length (i.e. unmutated) Orai1 subunits, and expressed this in HEK cells stably expressing STIM1. La3+-sensitive store-operated currents in these cells were evaluated in standard whole-cell patch clamp recordings using a Ca2+-free, Cs+-based (140 mM), internal (patch pipette) solution containing the potent InsP3-receptor agonist adenophostin A (2 μM). Under these conditions, maximal activation of store-operated Ca2+ currents occurred within approximately 85–120 s after achieving whole-cell conditions (see Supplementary Fig. S1), consistent with previous studies involving either Orai1 monomers, or concatenated Orai1 tetramers919. Examination of the resulting store-operated conductance measured in a Na+/Ca2+ based external solution ([Na+] = 140 mM, [Ca2+] = 10 mM) revealed a current-voltage relationship showing inward rectification, with clear outward currents observed at voltages above the reversal potential of +20 mV (Fig. 1a). Such outward currents are not seen with endogenous CRAC channel currents320, suggesting the presence of a significant permeability of the internal cation (Cs+). We next examined the effect of reducing extracellular Ca2+ from 10 mM to 0.1 mM, a concentration that is sufficient to render Ca2+ currents through CRAC channels to negligible levels, but is still sufficient, along with the presence of external Mg2+ (1.2 mM), to prevent any development of divalent-free currents321222324. This procedure resulted in only a modest reduction (less than 25%) in the inward current magnitude measured at −80 mV, and shifted the reversal potential to 0 mV (Fig. 1a). Together, these features are consistent with the presence of a significant permeability to the external Na+ ions, and only a modest overall contribution of Ca2+ ion flux. In addition, a significant (2-to-3-fold) increase in the outward currents recorded at positive potentials was seen, indicating that the reduction in extracellular Ca2+ concentration resulted in an increased permeability of the channel to Cs+ ions.


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

Thompson JL, Shuttleworth TJ - Sci Rep (2013)

Selectivity properties of expressed hexameric and tetrameric Orai1 concatamers.(a) Mean (±SE, n = 6) I/V curves for store-operated currents recorded between −100 mV and +80 mV in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM Na+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) The corresponding mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 tetramer. (c) Western blot showing the expressed FLAG-tagged concatamers running at their appropriate expected molecular masses (approximately 195 kDa for the hexamer, and 130 kDa for the tetramer).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Selectivity properties of expressed hexameric and tetrameric Orai1 concatamers.(a) Mean (±SE, n = 6) I/V curves for store-operated currents recorded between −100 mV and +80 mV in cells expressing the concatenated Orai1 hexamer in an external medium containing 140 mM Na+ and either 10 mM Ca2+, or 0.1 mM Ca2+. (b) The corresponding mean (±SE, n = 5) I/V curves for store-operated currents in cells expressing the concatenated Orai1 tetramer. (c) Western blot showing the expressed FLAG-tagged concatamers running at their appropriate expected molecular masses (approximately 195 kDa for the hexamer, and 130 kDa for the tetramer).
Mentions: Given the above contradictory findings, we chose to directly examine the functional properties of hexameric Orai1 channels in more detail. For this we generated a construct consisting of a concatenated hexameric assembly of full-length (i.e. unmutated) Orai1 subunits, and expressed this in HEK cells stably expressing STIM1. La3+-sensitive store-operated currents in these cells were evaluated in standard whole-cell patch clamp recordings using a Ca2+-free, Cs+-based (140 mM), internal (patch pipette) solution containing the potent InsP3-receptor agonist adenophostin A (2 μM). Under these conditions, maximal activation of store-operated Ca2+ currents occurred within approximately 85–120 s after achieving whole-cell conditions (see Supplementary Fig. S1), consistent with previous studies involving either Orai1 monomers, or concatenated Orai1 tetramers919. Examination of the resulting store-operated conductance measured in a Na+/Ca2+ based external solution ([Na+] = 140 mM, [Ca2+] = 10 mM) revealed a current-voltage relationship showing inward rectification, with clear outward currents observed at voltages above the reversal potential of +20 mV (Fig. 1a). Such outward currents are not seen with endogenous CRAC channel currents320, suggesting the presence of a significant permeability of the internal cation (Cs+). We next examined the effect of reducing extracellular Ca2+ from 10 mM to 0.1 mM, a concentration that is sufficient to render Ca2+ currents through CRAC channels to negligible levels, but is still sufficient, along with the presence of external Mg2+ (1.2 mM), to prevent any development of divalent-free currents321222324. This procedure resulted in only a modest reduction (less than 25%) in the inward current magnitude measured at −80 mV, and shifted the reversal potential to 0 mV (Fig. 1a). Together, these features are consistent with the presence of a significant permeability to the external Na+ ions, and only a modest overall contribution of Ca2+ ion flux. In addition, a significant (2-to-3-fold) increase in the outward currents recorded at positive potentials was seen, indicating that the reduction in extracellular Ca2+ concentration resulted in an increased permeability of the channel to Cs+ ions.

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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