کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1943959 | 1053169 | 2016 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Proteolytic cleavage in the S1–S2 linker of the Kv1.5 channel does not affect channel function Proteolytic cleavage in the S1–S2 linker of the Kv1.5 channel does not affect channel function](/preview/png/1943959.png)
• Proteinase K cleaves Kv1.5 channels at S1–S2 into two fragments.
• Co-IP experiments indicate that the N- and C-fragments do not associate.
• Proteinase K-cleavage of cell-surface channels does not affect Kv1.5 current.
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 (75 kDa) 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.
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Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes - Volume 1858, Issue 6, June 2016, Pages 1082–1090