At KC1 inhibits At AKT1 activation via its amino-terminal inhibitory domain

AKT1 is a plant Shaker-like, hyperpolarization-activated K ⁺ channel which plays a crucial role in K ⁺ absorption. Besides phosphorylation, AKT1 is subject to negative regulation by At KC1, a silent channel of the same family. Previous structural studies unveiled that AKT1 and At KC1 form 2:2 heterotetramer in purified samples. However, the structural analysis failed to offer more insight into the inhibition mechanism of AKT1 activation by At KC1. Here, inspecting the complex structure of AKT1- At KC1 reveals that a stable domain of At KC1 (residues 53 to 80), named inhibitory domain or I-domain, may inhibit AKT1 activation by stabilizing its depolarized, closed conformation. We confirmed this hypothesis with electrophysiological experiments. Interestingly, a single-point mutation (G315D) in At KC1 has been reported to abolish its ability to inhibit AKT1. We solved the structure of AKT1- At KC1(G315D) at a resolution of 2.8 Å which revealed an unexpected stoichiometry alteration between AKT1 and At KC1 from 2:2 to 3:1. This stoichiometry change further supports the hypothesis as we reason that single I-domain of At KC1 in the channel complex is insufficient to effectively inhibit channel activation. Our findings reveal the inhibition mechanism of At KC1 on AKT1 and offer insight into the regulatory mechanisms of hyperpolarization-activated channels.