Regulation of Kv1.5 by Extracellular Protease Calpain

Abstract

Atrial Fibrillation (AF) is a rapid, irregular heart rhythm caused by electrical signal disturbances in the upper chamber of the heart. The exact mechanism is complex and is currently incompletely understood. AF is the most common arrhythmia and affects between 2.3 – 4.3% of the general population. Furthermore, its incidence after cardiac surgery known as post-operative AF occurs in 20 – 40% of patients. The voltage gated potassium channel Kv1.5 is selectively expressed in the atria and conducts the repolarizing current IKur. The mechanisms underlying AF are not completely understood, but dysfunction of Kv1.5 can cause AF, and pharmacological intervention targeting Kv1.5 is a recognized strategy for the treatment of AF. Previous research from our lab showed that the Kv1.5 channel is cleaved by a fungal enzyme Proteinase K (PK), which cleaves the mature, cell-surface Kv1.5 protein, leading to an increase in the Kv1.5 current (IKv1.5). Such alterations to the function of Kv1.5 would change the duration of the atrial action potential, creating pathophysiological substrate for AF. In the present study, mutagenesis, patch clamp, and Western blot were used to investigate if there are endogenous proteases that exert an effect like PK, leading to an increase in IKv1.5. The results show that calpain, a cysteine protease that is ubiquitously expressed in mammals and upregulated in inflammatory conditions, cleaves the mature Kv1.5 protein, and results in an increased IKv1.5. Based on the size of the fragments resulting from the cleavage, we identified a potential cut site located between residues 282-300 in the extracellularly exposed S1-S2 linker of the channel. Removal of these residues abolished calpain-mediated cleavage of Kv1.5 and prevented the increase in IKv1.5. Our previous work has shown that Kv1.5 is constantly undergoing endocytosis via Src targeting to the N-terminus of the channel. Indeed, deletion of the Src target-site in the N-terminus completely abolished calpain-mediated increase in IKv1.5. The membrane expression level of Kv1.5 is a balance between endogenous Src-mediated internalization and new channel insertion. Src mediates Kv1.5 internalization by targeting the N-terminus of channel. Following cleavage, the C-terminus fragment is separated from the N-terminus fragment, and thus is not internalized by Src and remains on the cell surface. Since the C-terminus fragment contains the pore-forming domain, it continues conducting a current. Accumulation of these fragments on the cell surface, in addition to the insertion of new channels being would result in an increased overall current density. These findings offer a novel mechanistic explanation for the increase in current following cleavage by proteases and provide insight into the arrhythmogenic effects of proteases released during post-operative inflammation.