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Polyamine Interaction Sites Located by Combined Channel and Ligand Mutagenesis
2 Department of Physiology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada
3 Département de Médecine Moléculaire, Institut Pasteur, 75724 Paris, Cedex 15, France
4 Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Institut du Thorax, 44035 Nantes, France
5 Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106
6 Friedrich Schiller University Jena, Institute of Physiology II, 07743 Jena, Germany
Address correspondence to Colin G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Fax: 314-362-7463; email: cnichols{at}cellbio.wustl.edu
Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.
Key Words: inward rectifier • spermine • diamine • rectification • selectivity filter
Abbreviations used in this paper: DA9, 1,9 diamino-nonane; DAn, (1,n)-diaminoalkanes; WT, wild type.
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