Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans

Heiner Splitt; Dirk Meuser; Ilya Borovok; Michael Betzler; Hildgund Schrempf

doi:10.1016/S0014-5793(00)01429-0

Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans

Heiner Splitt, Dirk Meuser, Ilya Borovok, Michael Betzler, Hildgund Schrempf^*

^*Corresponding author for this work

Osnabrück University

Research output: Contribution to journal › Article › peer-review

48 Scopus citations

Abstract

Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K⁺ channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity. Copyright (C) 2000 Federation of European Biochemical Societies.

Original language	English
Pages (from-to)	83-87
Number of pages	5
Journal	FEBS Letters
Volume	472
Issue number	1
DOIs	https://doi.org/10.1016/S0014-5793(00)01429-0
State	Published - 21 Apr 2000
Externally published	Yes

Keywords

Electrophysiology
Mutant KcsA
Tetramerization
Thermal stability

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10.1016/S0014-5793(00)01429-0

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title = "Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans",

abstract = "Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K+ channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity. Copyright (C) 2000 Federation of European Biochemical Societies.",

keywords = "Electrophysiology, Mutant KcsA, Tetramerization, Thermal stability",

author = "Heiner Splitt and Dirk Meuser and Ilya Borovok and Michael Betzler and Hildgund Schrempf",

note = "Funding Information: M. Heggemann helped with the cloning experiments and D. M{\"u}ller with the propagation of phages. We thank R. Wagner for providing equipment for electrophysiological studies, for his interest in our work and for prolific discussions. M. Lemme supported the writing of the manuscript. D. Meuser, I. Borovok (guest scientist) and H. Splitt have been financed by the Graduiertenkolleg at the University of Osnabr{\"u}ck, the Minerva foundation, Munich, and the Sonderforschungsbereich 431 (project D5, H. Schrempf) of the University of Osnabr{\"u}ck, respectively; through the SFB 431 also the costs for the experiments and studies have been covered.",

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doi = "10.1016/S0014-5793(00)01429-0",

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T1 - Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans

AU - Splitt, Heiner

AU - Meuser, Dirk

AU - Borovok, Ilya

AU - Betzler, Michael

AU - Schrempf, Hildgund

N1 - Funding Information: M. Heggemann helped with the cloning experiments and D. Müller with the propagation of phages. We thank R. Wagner for providing equipment for electrophysiological studies, for his interest in our work and for prolific discussions. M. Lemme supported the writing of the manuscript. D. Meuser, I. Borovok (guest scientist) and H. Splitt have been financed by the Graduiertenkolleg at the University of Osnabrück, the Minerva foundation, Munich, and the Sonderforschungsbereich 431 (project D5, H. Schrempf) of the University of Osnabrück, respectively; through the SFB 431 also the costs for the experiments and studies have been covered.

PY - 2000/4/21

Y1 - 2000/4/21

N2 - Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K+ channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity. Copyright (C) 2000 Federation of European Biochemical Societies.

AB - Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K+ channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity. Copyright (C) 2000 Federation of European Biochemical Societies.

KW - Electrophysiology

KW - Mutant KcsA

KW - Tetramerization

KW - Thermal stability

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JF - FEBS Letters

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ER -

Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans

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Keywords

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