Grb10 adaptor decreases TrkB-induced phosphorylation of Kv1.3 while nShc adaptor increases channel phosphorylation. (A) HEK 293 cells were transiently transfected with cDNA encoding Kv1.3 (K) or Kv1.3 plus TrkB kinase (T) with either Grb10 (G) or nShc (S). A dead TrkB kinase (D) where the catalytic β chain was truncated (see Fig. 1, TrkBTrunc) was also incorporated into the transfection design (KDS). Cells were stimulated with vehicle (-) or BDNF (+), and lysates were prepared and separated by SDS-PAGE as in Fig. 4. Upper panel shows representative expression bands for three such experiments in which the nitrocellulose was probed with αAU13 to recognize Kv1.3 channel protein (anti-Kv1.3). Note increase in Kv1.3 expression with TrkB co-expression and decreased Kv1.3 expression in the Grb10 co-transfection condition. Lower panel lysates were IP with αAU13 (anti-Kv1.3) and then nitrocellulose was probed with an antiserum that recognizes tyrosine phosphorylated proteins (anti-4G10) to quantify the degree of channel phosphorylation (pKv1.3). The Mr for Kv1.3 ranges from 55 to 72 kDa depending upon extent of phosphorylation (< Kv1.3). (B) Same experimental paradigm, protocol, notation and sample size as in (A) but substitution of myc-tagged Kv1.3 (mK) to quantify fraction of pKv1.3 at the surface membrane as opposed to total cellular channel protein. HEK 293 cells were transiently transfected with cDNA encoding myc-tagged Kv1.3 (mK) plus nShc (S) and wildtype TrkB (T) or mutantTrkB kinase (TShc-) lacking the Shc binding site. Upper panel lysates were IP with αmyc and then nitrocellulose was probed with α4G10 (anti-4G10) to quantify degree of channel phosphorylation (pKv1.3). Note loss of pKv1.3 in lanes 5–6 (upper panel) without the Shc binding site, despite expression of the channel in both whole cell fraction (middle panel) and membrane fraction (lower panel). (C) Bar graph plot of the mean ± S.E.M. normalized immunodensity values of the pKv1.3 labeled band for experiments demonstrated in A. Pixel density ratios (dashed line, 1.0 no difference) were generated for BDNF stimulated (+) conditions by normalizing the pixel density for a transfection condition (KT, KTG, KTS, or KDS) to that of control K transfection condition (K) within a single autoradiographic film to eliminate variability introduced by differential exposure times. * = significantly different compared with control K transfection condition, by Student's t-test, arc sine transformation for percentage data, α ≤ 0.05.