Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

The Journal of General Physiology
Yuichiro Fujiwara, Yoshihiro Kubo

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 b...Continue Reading

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Citations

Jan 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·Aldo A Rodríguez-MenchacaJosé A Sánchez-Chapula
Nov 8, 2008·Proceedings of the National Academy of Sciences of the United States of America·Katsuhiro Nagatomo, Yoshihiro Kubo
Jul 22, 2009·The Journal of Biological Chemistry·Masanori OsawaIchio Shimada
Oct 11, 2011·The Journal of Biological Chemistry·Atsushi InanobeYoshihisa Kurachi
Jan 10, 2013·The Journal of Biological Chemistry·Harley T KurataColin G Nichols
Nov 22, 2013·The Journal of Biological Chemistry·Lejla ZubcevicStephen J Tucker
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Jun 27, 2007·The Journal of General Physiology·Indra Schroeder, Ulf-Peter Hansen
Dec 31, 2008·The Journal of General Physiology·Yuichiro FujiwaraYoshihiro Kubo
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Apr 28, 2010·The Journal of General Physiology·Harley T KurataColin G Nichols
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Methods Mentioned

BETA
transfection
PCR

Software Mentioned

KyPlot
Sliders
Clampfit
pCLAMP

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