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Fig. 3. Sequence information in the Kir2.1 C-terminus is both necessary and sufficient to promote Golgi-to-plasma membrane transport. (A) Schematic drawing of Kir2.1/Kir2.4 chimeric constructs. Kir2.4-2.1(N): _1-77Kir2.4 was substituted by _1-69Kir2.1; Kir2.4-2.1(C): _212-439Kir2.4 was substituted by _204-428Kir2.1; Kir2.4-int(2.1): _241-260Kir2.4 was replaced by _233-252Kir2.1; Kir2.1-int(2.4): _233-252Kir2.1 was replaced by _241-260Kir2.4. (B) The C-terminal domain of Kir2.1 contains critical sequence information that is sufficient to increase Kir2.4 surface expression. Representative confocal images of OK cells expressing extracellularly HA-tagged _GFPKir2.4 and _GFPKir2.4-2.1(C), processed for -HA immunocytochemistry without membrane permeabilization (TX: Triton X-100). (C) A stretch of 20 amino acids of Kir2.1 (_233-252Kir2.1) is sufficient to increase surface expression of Kir2.4 and necessary for efficient surface expression of Kir2.1. Representative confocal images of OK cells expressing the indicated extracellularly HA-tagged constructs, processed for detecting their surface expression. Total protein expression of the various constructs is similar as shown in -GFP immunoblot analysis below. (D) Quantification of the surface expression experiments in (C). (E) The Kir2.1 trafficking determinant actively promotes Golgi-to-plasma membrane transport. Substitution of _233-252Kir2.1 by a flexible glycine-serine linker peptide (Kir2.1-GS) reduces surface expression of Kir2.1, whereas Kir2.4 surface expression remains unaffected by homologous substitution of _241-260Kir2.4 by the linker peptide (Kir2.4-GS). *Statistically significant when compared with respective controls (P<0.01; unpaired Student's t-test; see Materials and Methods).

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