Investigating the Dynamics of the KCNN4 Channel: From the Determination of the Complete K⁺ Permeation Pathway Across the Channel to Its Opening by PIP2

KCNN4 is a calcium (Ca)-activated potassium channel for which Ca²⁺ sensitivity is conferred by calmodulin (CaM) that constitutively binds to the channel. Until the main part of the structure bound to CaM has been resolved, <i>in silico</i> studies had used homology models derived from the well characterized transmembrane domain of other K⁺ channels, limiting the functional investigation to this particular region. Thus, how the regulatory domains of KCNN4 communicate with each other and where the possible gates are located across the complete structure are still not well understood. Here we present for the first time results obtained from the investigation of full-length models of the channel in different conformational states and molecular contexts using classical all-atom molecular dynamic simulations. The simulations covered two activated states (open and closed) and a preactivated state of the channel embedded in a simple membrane model and a model of red blood cell membrane, where the channel is functionally expressed <i>in vivo</i>. Surprisingly, the intracellular domain was refractory to the entrance of K⁺, whatever the state of the channel was, allowing the K⁺ ions to enter and exit the channel only through two newly identified restrained diffusion spots. Inside the channel, the K⁺ flux was controlled by the V282 residue closing the pore region when the CaM N-lobes were not bound. This flux was compatible with the passage of fully or partially hydrated K⁺, depending on the opening level. Finally, the presence of phosphatidylinositol-4,5-bisphosphate (PIP2), a well-known K⁺-channel modulator, in a putative binding site of KCNN4 clearly facilitated the opening of the V282 restriction. Thus, in addition to the elucidation of the possible complete K⁺ permeation pathway throughout KCNN4, our results confirmed the direct activatory role of PIP2, associated with the channel opening, and provide a first insight into the architecture and the behavior of the complete intracellular region of KCNN4.