title: Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
abstract: Kv1.5 channels mediate the ultra-rapidly activating delayed rectifier potassium current (IKur), which is important for atrial repolarization. It has been shown that cell-surface Kv1.5 channels are sensitive to cleavage by the extracellular serine protease, proteinase K (PK). Here, we investigated the effects of extracellular proteolytic digestion on the function of Kv1.5 channels stably expressed in HEK 293 cells. Our data demonstrate that PK treatment cleaved mature membrane-bound (75kDa) Kv1.5 channels at a single locus in the S1-S2 linker, producing 42-kDa N-terminal fragments and 33-kDa C-terminal fragments. Interestingly, such PK treatment did not affect the Kv1.5 current (IKv1.5) recorded using the whole-cell patch clamp technique. Analysis of cell-surface proteins isolated using biotinylation indicated that the PK-generated N- and C-terminal fragments were both present in the plasma membrane. Co-immunoprecipitation (co-IP) experiments indicated that the N- and C-terminal fragments are no longer associated after cleavage. Furthermore, following PK digestion, the N- and C-fragments degraded at different rates. PK is frequently used as a tool to analyze cell-surface localization of membrane proteins, and cleavage of cell-surface channels has been shown to abolish channel function (e.g. hERG). Our data, for the first time, demonstrate that cleavage of cell-surface channels assessed by Western blot analysis does not necessarily correlate with an elimination of the channel activities.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hogan-Cann
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
authors.forename: Wentao
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
authors.forename: Tonghua
authors.initials: T
authors.lastname: Yang
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: shetuan.zhang@queensu.ca.
authors.forename: Shetuan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D051666:Kv1.5 Potassium Channel / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D059748:Proteolysis
chemical_list: C496981:KCNA5 protein, human; D051666:Kv1.5 Potassium Channel
keywords: Cell-surface protein; Electrophysiology; Kv1.5; Trafficking; Voltage-gated potassium channel