A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating.
Store-operated Ca(2+) entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca(2+) sensors with calcium release-activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20-amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.
Center for Biomedical Research, The Queen's Medical Center, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96813, USA.
Annette
A
Lis
Susanna
S
Zierler
Christine
C
Peinelt
Andrea
A
Fleig
Reinhold
R
Penner
D001896:Boron Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D014162:Transfection
D001896:Boron Compounds; D015220:Calcium Channels; D008565:Membrane Proteins; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D008239:Lysine; D002118:Calcium