Interplay of Orai1-Loop3 with Extracellular Ca2+ Binding Sites in Loop1 Controls Crac Channel Activity
| Autor: | Barbora Svobodova, Irene Frischauf, Anna Hochreiter, Michael Deix, Rainer Schindl, Christoph Romanin, Vasilina Zayats, Rüdiger Ettrich |
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| Rok vydání: | 2014 |
| Předmět: |
0303 health sciences
Chemistry ORAI1 Protein subunit Biophysics Nanotechnology STIM1 Sequence alignment Coupling (electronics) 03 medical and health sciences Molecular dynamics 0302 clinical medicine Extracellular Membrane channel sense organs 030217 neurology & neurosurgery 030304 developmental biology |
| Zdroj: | Biophysical Journal. 106(2) |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2013.11.1827 |
| Popis: | Ca2+ release-activated Ca2+ (CRAC) channels are a major pathway for Ca2+ entry in a vast majority of cell types. The identification of the ER Ca2+ sensor STIM1 and the plasma membrane channel subunit Orai1 lead to a substantial progress in our understanding of molecular structures underlying the unique permeation properties of CRAC channels. Nevertheless, Ca2+ coordination at the pore entrance and the role of the extracellular loop1 and loop3 of hOrai1 proteins still remain rather unclear.Based on a recent crystal structure of the drosophila Orai channel we generated an all-atom model of human Orai1 including the missing extracellular loops. Using a combined approach of patch-clamp, cysteine-crosslinking and molecular dynamics simulations we investigated the pore entrance of hOrai1 channels. Our experiments showed that the extracellular loop3 of hOrai1 exhibited a high degree of crosslinking along the whole loop compatible with a remarkable flexibility. Crosslinking studies between loop1 and loop3 demonstrated close proximity, suggesting a movement of loop3 affecting the pore entrance. Molecular dynamics simulations indicated that Ca2+ ions bind to the extracellular aspartates located in the first loop. Further, an interaction of the third loop with these negatively charged amino acids seems to compete with the Ca2+ ions, thereby reducing CRAC channel activity. Sequence alignment of Orai1 isoforms identified that the essential residues for hOrai1 loop1-loop3 coupling are conserved in higher mammals.With this work we suggest a pivotal/regulatory role of the third loop in modulating hOrai1 function. Via movement and subsequent coupling of loop3 to aspartates in the first loop it may regulate the Ca2+ sink at the pore entrance and thus permeation through CRAC channels.This work was supported by the Austrian Science Foundation (FWF): P22747, P26067 (R.S.), P25172 (C.R.), V286 (I.F.). |
| Databáze: | OpenAIRE |
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