Aberrant TMPRSS6-Protease Regulation of Disease Mutant HCN4-KCNE1 Channel Complex Depends on the KCNE1-G38S...

The sinoatrial node pacemaker channel HCN4 plays a central role in cardiac automaticity, and disease-associated variants can predispose to atrial arrhythmias. Here, we investigated the functional interplay between the HCN4 variant P883R and the potassium channel β-subunit KCNE1, focusing on the common atrial fibrillation-associated KCNE1 variant G38S and its regulation by the iron-induced serine protease TMPRSS6. Electrophysiological analyses revealed that HCN4-P883R decreases net HCN4 currents I_f, consistent with impaired automaticity. Co-expression of KCNE1, either wild-type or polymorphic KCNE1-G38S, restored functional properties of the mutant channel, indicating that KCNE1 is a key modulator of HCN4 activity. Importantly, TMPRSS6-mediated proteolytic processing of KCNE1 reduced HCN4 currents, whereas HCN4 expressed alone was insensitive to TMPRSS6, identifying KCNE1 as the direct regulatory target. Notably, KCNE1-G38S altered the HCN4-KCNE1 complex to TMPRSS6-dependent downregulation, resulting in a reduced suppression of HCN4-P883R-mediated currents compared with wild-type KCNE1. Mechanistically, differential TMPRSS6 cleavage depended on the membrane positioning of the KCNE1-³²RRSPRSS³⁸ motif. These findings reveal a protease-dependent buffering mechanism that counteracts HCN4 loss-of-function and establish TMPRSS6 as a molecular switch controlling pacemaker activity in a KCNE1 genotype-dependent manner. This dynamic regulatory framework may contribute to the phenotypic variability of sinoatrial node dysfunction and atrial fibrillation.