Screening Of Negative Charges By Ca2+ In The Turret Region Controls Kv7.1 Inactivation Gating And Is Regulated By Pip2 And Calmodulin
BIOPHYSICAL JOURNAL(2019)
摘要
Inactivation is an intrinsic property of numerous voltage-gated K+ (Kv) channels and can occur by N-type or/and C-type mechanisms. While fast N-type inactivation involves the inner pore occlusion by N-terminal peptide domains of α and β subunits, C-type inactivation is suggested to involve structural rearrangements in the outer pore leading to a loss of K+ coordination sites in the selectivity filter. In Kv7.1 channels, inactivation is invisible macroscopically and does not exhibit the hallmarks of N- and C-type mechanisms. However, Kv7.1 inactivation is revealed by hooked tail currents, which reflect the recovery from an inactivation state. We show that removal of external Ca2+ increased the activation kinetics and produced a large voltage-dependent inactivation of Kv7.1 channels. Increasing external Ca2+ suppresses inactivation gating with an EC50 of 1.5 μM. While Sr2+ and Cd2+ mimicked the effects of Ca2+, other divalent cations like Mg2+ and Mn2+ were ineffective. External K+ (50 mM) did not prevent the inactivation evoked in Ca2+-free external solutions suggesting a mechanism different from C-type inactivation. External acidification or introduction in the pipet solution of calcified calmodulin or PIP2 slowed down the activation kinetics and precluded inactivation gating evoked in Ca2+-free external solutions. Experimental data and kinetic modeling indicated that Kv7.1 channels exhibit two distinct inactivation states. Mutagenesis studies and structural modeling suggest that external Ca2+ ions act to screen the negative charges of neighboring glutamate and aspartate residues located respectively, in the turret and filter entrance of the channel pore. Our results reveal a new mechanism whereby external Ca2+ exquisitely controls inactivation gating of a Kv channel that is allosterically modulated by PIP2 and calcified calmodulin at the inner face of the channel transmembrane core.
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关键词
negative charges,inactivation,turret region
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