Increased phosphorylation of Kv1.4 subunits in Kv1.4/Kv1.5
heteromultimers mediated by the interaction of Src with the Kv1.5
proline-rich SH3 domain ligand sequence. (a) Stable
association of Src PTK with Kv1.4/Kv1.5 heteromultimeric channels
depends on the interaction of Src with the proline-rich 2xRPLPPLP
sequence of Kv1.5. Combinations of Kv1.4, Kv1.5-Tag, Kv1.5-ΔPro-Tag,
and c-Myc epitope-tagged catalytically impaired Src
(SrcCI-Tag) were transfected into HEK 293 cells, followed
by Western blot of α−Kv1.4 immunoprecipitates of cell lysates.
SrcCI coimmunoprecipitates with Kv1.4/Kv1.5
heteromultimers, but fails to coimmunoprecipitate with either Kv1.4
homomultimers or Kv1.4/Kv1.5-ΔPro heteromultimers.
(b) Increased phosphorylation of Kv1.4 subunits by Src
in Kv1.4/Kv1.5 heteromultimers depends on the Kv1.5 2xRPLPPLP Src SH3
domain ligand. Cell lysates from a were
immunoprecipitated with α−Kv1.5-Tag mAb and analyzed by Western
blot. The Kv1.4 subunits coassembled with Kv1.5 exhibit greater
tyrosine phosphorylation than the Kv1.4 subunits coassembled with
Kv1.5-ΔPro. (c) Quantitative comparison of Kv1.4
subunit phosphorylation in cells transfected with combinations of
Kv1.4, Kv1.5-Tag, Kv1.5-ΔPro-Tag, and SrcCI-Tag. Kv1.4
phosphorylation was quantitatively analyzed as in Fig.
2d, but with relative density defined in relation to the
normalized density observed for the
Kv1.4/Kv1.5-Tag/SrcCI-Tag transfection condition
(mean ± SEM, n = 4; **,
P < 0.001 by paired t test).