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Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1.
J Gen Physiol. 2006 Apr; 127(4):401-19.JG

Abstract

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.

Authors+Show Affiliations

Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan.yuichiro@nips.ac.jpNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16533896

Citation

Fujiwara, Yuichiro, and Yoshihiro Kubo. "Functional Roles of Charged Amino Acid Residues On the Wall of the Cytoplasmic Pore of Kir2.1." The Journal of General Physiology, vol. 127, no. 4, 2006, pp. 401-19.
Fujiwara Y, Kubo Y. Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1. J Gen Physiol. 2006;127(4):401-19.
Fujiwara, Y., & Kubo, Y. (2006). Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1. The Journal of General Physiology, 127(4), 401-19.
Fujiwara Y, Kubo Y. Functional Roles of Charged Amino Acid Residues On the Wall of the Cytoplasmic Pore of Kir2.1. J Gen Physiol. 2006;127(4):401-19. PubMed PMID: 16533896.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1. AU - Fujiwara,Yuichiro, AU - Kubo,Yoshihiro, Y1 - 2006/03/13/ PY - 2006/3/15/pubmed PY - 2006/8/11/medline PY - 2006/3/15/entrez SP - 401 EP - 19 JF - The Journal of general physiology JO - J Gen Physiol VL - 127 IS - 4 N2 - It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance. SN - 0022-1295 UR - https://www.unboundmedicine.com/medline/citation/16533896/Functional_roles_of_charged_amino_acid_residues_on_the_wall_of_the_cytoplasmic_pore_of_Kir2_1_ L2 - https://rupress.org/jgp/article-lookup/doi/10.1085/jgp.200509434 DB - PRIME DP - Unbound Medicine ER -