Proteolytic Cleavage of the Kv1.5 Channel in the S1-S2 Linker Does Not Affect Channel Function

Abstract

Atrial fibrillation (AF), the most prevalent human cardiac arrhythmia, is characterized by rapid and disorderly electrical activity in the atria of the heart. Kv1.5 channel mediated ultra-rapidly activating delayed rectifier potassium current (IKur) is critical for timely and adequate atrial repolarization. Since cardiac IKur is specific to the atria, biomedical research surrounding Kv1.5 poses significant promise for developing clinical strategies to treat AF. In fact, loss-of-function mutations in KCNA5 (encoding Kv1.5) have been identified in patients with AF. Importantly, common pathologies, such as selective atrial ischemia, are capable of stimulating the onset of AF. A well-documented consequence of ischemia is a substantial increase in proteolytic enzyme activities. In this regard, it has been reported that cell-surface Kv1.5 channels are sensitive to cleavage by extracellular proteases, such as proteinase K (PK). In this study, we further examined the effects of extracellular proteases on the function and expression profile of Kv1.5 channels stably expressed in HEK 293 cells. Our results demonstrate that PK cleaves membrane-bound mature (75-kDa) Kv1.5 channels at a single locus in the external S1-S2 linker, yielding 42-kDa N-terminal and 33-kDa C-terminal fragments. Contrary to our expectations, whole-cell voltage clamp analysis showed that PK treatment did not affect Kv1.5 current (IKv1.5). Examination of plasma membrane proteins isolated via biotinylation indicated that the N- and C-terminal Kv1.5 fragments were both present and stable on the cell-surface. Co-immunoprecipitation (co-IP) studies following PK cleavage suggest that the two Kv1.5 fragments do not associate. Moreover, the PK-generated N- and C-terminal fragments degraded at different rates. These findings indicate that the C-terminal fragment of Kv1.5 (S2-S6, pore-containing) may be sufficient for current conduction. Our data raises the possibility that cleavage of cell-surface ion channels, assessed by Western blot analysis, does not necessarily result in a loss of channel activities. This novel insight into the Kv1.5 structure-function relationship may be indicative of an inherent protective mechanism for Kv1.5 channel function.