CaT1 knock-down strategies fail to affect CRAC channels in mucosal-type mast cells.
CaT1, the calcium transport protein 1 encoded by TRPV6, is able to generate a Ca(2+) conductance similar but not identical to the classical CRAC current in mucosal-type mast cells. Here we show that CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited either by expression of various dominant negative N-terminal fragments of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense suppression. By contrast, the endogenous CRAC current of the mast cells was unaffected by CaT1 antisense and siRNA knockdown but markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant negative N-CaT1 fragments. Inhibition of CRAC current was not an unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents of the mast cells were not significantly affected by these N-CaT1 fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived currents in mast cells, but failed to inhibit CRAC currents. Thus, the structural requirements of rCaT N-terminal fragments for inhibition of rCaT1 and CRAC channels are different. These results together with the lack of CaT1 antisense and siRNA effects on currents render it unlikely that CaT1 is a component of native CRAC channels in mast cells. The data further demonstrate a novel strategy for CRAC current inhibition by an N-terminal structure of CaT1.
Kahr
Heike
H
Institute for Biophysics, University of Linz, A-4040 Linz, Austria.
Schindl
Rainer
R
Fritsch
Reinhard
R
Heinze
Barbara
B
Hofbauer
Michael
M
Hack
Marlene E
ME
Mörtelmaier
Manuel A
MA
Groschner
Klaus
K
Peng
Ji-Bin
JB
Takanaga
Hitomi
H
Hediger
Matthias A
MA
Romanin
Christoph
C
D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D016257:Fura-2; D006801:Humans; D008407:Mast Cells / Q000378:metabolism*; D009092:Mucous Membrane / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D034741:RNA, Small Interfering / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071176:Signaling Lymphocytic Activation Molecule Family; D050916:TRPV Cation Channels; D014162:Transfection
D015220:Calcium Channels; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; C448987:SLAMF7 protein, human; D000071176:Signaling Lymphocytic Activation Molecule Family; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D002118:Calcium; D016257:Fura-2