TY - JOUR
T1 - Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel
AU - Hirano, Minako
AU - Ide, Toru
N1 - Funding Information:
This work was supported by JSPS KAKENHI Grant Number 25840055 and 15K07035 .
Publisher Copyright:
© 2018
PY - 2019/1
Y1 - 2019/1
N2 - KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.
AB - KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.
KW - Inactivation
KW - KcsA
KW - Potassium channel
UR - http://www.scopus.com/inward/record.url?scp=85050666585&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85050666585&partnerID=8YFLogxK
U2 - 10.1016/j.bbamem.2018.07.011
DO - 10.1016/j.bbamem.2018.07.011
M3 - Article
C2 - 30053405
AN - SCOPUS:85050666585
VL - 1861
SP - 220
EP - 227
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
SN - 0005-2736
IS - 1
ER -