Modifications of alamethicin ion channels by substitution of Glu-7 for Gln-7.
To evaluate the role of charged residues facing a pore lumen in stability of channel structure and ion permeation, we studied electrical properties of ion channels formed by synthesized native alamethicins (Rf50 (alm-Q7Q18) and Rf30 (alm-Q7E18)) and their analogs with Glu-7 (alm-E7Q18 and alm-E7E18). The single-channel currents were measured over a pH range of 3.5 to 8.7 using planar bilayers of diphytanoyl PC. The peptides all showed multi-level current fluctuations in this pH range. At pH 3.5 the channels formed by the four peptides were similar to each other irrespective of the side chain differences at positions 7 and 18. The ionization of Glu-7 (E7) and Glu-18 (E18) above neutral pH reduced the relative probabilities of low-conductance states (levels 1 and 2) and increased those of high-conductance states (levels 4-6). The channel conductance of the peptides with E7 and/or E18, which was distinct from that of alm-Q7Q18, showed a marked pH-dependence, especially for low-conductance states. The ionization of E7 further reduced the stability of channel structure, altered the current-voltage curve from a superlinear relation to a sublinear one, and enhanced cation selectivity. These results indicate that ionized E7 strongly influences the channel structure and the ion permeation, in contrast to ionized E18.
Asami
Koji
K
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. asami@tampopo.kuicr.kyoto-u.ac.jp
Okazaki
Takashi
T
Nagai
Yasuaki
Y
Nagaoka
Yasuo
Y
D000408:Alamethicin / Q000737:chemistry*; D000595:Amino Acid Sequence; D000838:Anions; D055592:Biophysical Phenomena; D001703:Biophysics; D002713:Chlorine / Q000737:chemistry; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology*; D007477:Ions; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011188:Potassium / Q000737:chemistry
D000838:Anions; D007476:Ionophores; D007477:Ions; D008051:Lipid Bilayers; D010455:Peptides; D005973:Glutamine; D000408:Alamethicin; D018698:Glutamic Acid; D002713:Chlorine; D011188:Potassium