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It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

KCNQ1 Mutations in Early-Onset Lone AF Introduction Atrial fibrillation (AF) is the most frequent cardiac arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutation in KCNQ1, the gene encoding the pore-forming α-subunit of the IKs channel (KV7.1), was the first ion channel dysfunction to be associated with familial AF. We hypothesized that early-onset lone AF is associated with a high prevalence of mutations in KCNQ1. Methods and Results We bidirectionally sequenced the entire coding sequence of KCNQ1 in 209 unrelated patients with early-onset lone AF (

The voltage-gated potassium channel KV7.1 is regulated by non-pore forming regulatory KCNE β-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs. However, where the subunits assemble and which of the subunits determines localization of the IKs-complex has not been unequivocally resolved yet. We employed trafficking-deficient KV7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV7.1 targets the IKs-complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV7.1/KCNE complexes.

Aim The voltage-gated potassium channel KV11.1 is the molecular basis for the IKr current which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesised that the post-translational modification sumoylation finetunes channel activity. Methods The functional effects of sumoylation on KV11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analyse of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. Results Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93, and K116) in the PAS-domain as the putative SUMO-targets. Conclusion This study indicates KV11.1 as a sumoylation target, and offers the three main targets; K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain. This article is protected by copyright. All rights reserved.