A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model

Abstract

Small-conductance Ca2+-activated K+ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca2+-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca2+/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca2+ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP2-interacting residue K405, and subsequently enhanced Ca2+ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca2+ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca2+ with increased activity in vivo.

Hydrophobic interactions between the HA helix and S4‐S5 linker modulate apparent Ca2+ sensitivity of SK2 channels

“…Using site-directed mutagenesis and electrophysiological recordings, we found that decreasing hydrophobicity by the F410A mutation led to the hyposensitivity of SK2 channels to Ca 2+ , whereas increasing hydrophobicity by V407F mutation caused hypersensitivity of SK2 channels to Ca 2+ . 35 Limited by the structural information available back then, we were only able to identify M411 as a residue that could interact with the ectopic phenylalanine in the hyperactive V407F mutant channels. With a cryo-EM structure of the human SK4 channel published in 2018 36 as the template, we generated a homology model of the rat SK2 channel ( Figure 1A and Figure S1) as described previously, 35 which allowed us to take a fresh look at the cognate V407 residue.…”

“…The homology model of the rat SK2 channel was described in our previously report. 35 The WT or N293A/V407F mutant SK2 channels were immersed in an explicit lipid bilayer of POPC, POPE, POPS and cholesterol with molecular ratio of 25:5:5:1, 40 and a water box in 168.1Åx165.6Åx140.0Å dimension by using the CHARMM-GUI Membrane Builder webserver (http://www.charm m-gui.org/?doc=input/ membrane). 150mM KCl and extra neutralizing counter ions were added into the systems.…”

“…Both V407 and M411 are located at the interface between the HA helix and S 45 A-S 45 B helices ( Figure 1B). With the previous knowledge on the relationship between the hydrophobicity of HA helix and channel apparent Ca 2+ sensitivity, 35 we first investigated the large hydrophobic/aromatic residues in the S4-S5 linker, using an alanine-scanning mutagenesis strategy. The effect of substituting for alanine at the isoleucine and phenylalanine residues on the Ca 2+ -dependent channel activation was examined using inside-out patch-clamp recordings on the mutant channels heterologously expressed in HEK293 cells ( Figure S2).…”

“…[32][33][34] We recently identified that a valine to phenylalanine mutation in this region (V407F) results in a ~ 6-fold increase in apparent Ca 2+ sensitivity of SK2 channels. 35 A recent cryo-EM study of the SK4 channel subtype demonstrated that the CaM interacts with the linker between S4 and S5 transmembrane domains (S4-S5 linker), in addition to the CaMBD. 36 Utilizing this cryo-EM structure of human SK4 channels (PDB code: 6CNN) as a template, we generated a homology model of the rat SK2 channels as previously described ( Figure 1A).…”

“…36 Utilizing this cryo-EM structure of human SK4 channels (PDB code: 6CNN) as a template, we generated a homology model of the rat SK2 channels as previously described ( Figure 1A). 35 The CaMBD forms two α-helices, HA and HB, while the S4-S5 linker consists of two α-helices, S 45 A and S 45 B ( Figure 1B). The HA helix from one channel subunit (green), the S 45 A and S 45 B helices from a neighbouring channel subunit (yellow) and CaM (blue) closely interact with each other.…”