Figure 2: CaM binds to TRPA1 depending on Ca2+.
From: Calmodulin is responsible for Ca2+-dependent regulation of TRPA1 Channels
(A) HEK293 cell lysates expressing TRPA1-V5 or TRPV1-V5 (left panel) or non-transfected HEK293 cell lysates (lane 1 on the right panel) were pulled down by CaM-agarose with or without Ca2+ (1 mM) followed by detection with anti-V5. Total cell lysate (TCL) shows the expression of TRPA1 and TRPV1. Molecular weight is shown on the right, similar to all other blots. (B) Different thermo-TRP ion channels were pulled down by CaM-agarose in 2 mM Ca2+. The bottom blot shows the expression of individual TRP channels in total cell lysate (TCL). Multiple bands of TRP channels are caused by channel glycosylation56. The relative binding of TRPA1, TRPV1, 2, 3, 4 and TRPM8 to CaM (normalized by their total input) is 22.5, 0.3, 0.8, 0.2, 1.4 and 0.9, respectively. (C,D) CaM pull down assay shows the interaction of CaM with TRPA1 in nanomolar ranges of Ca2+ (C) and in different cations (1 mM each) (D). The bottom blots show equal expression of TRPA1 in different groups. (E) HA-tagged CaM or related mutants co-expressed with TRPA1-V5 in HEK293 cells as indicated, was immuno-precipitated (IP) by anti-HA in 2 mM Ca2+ followed by detection with anti-V5 (top blot). The bottom blot shows total input TRPA1 proteins. (F) Summary of relative TRPA1 binding to CaM (normalized by total TRPA1 proteins shown at the bottom blot in E) from similar experiments to those in E (n = 3).
