Increased resistance to extracellular cation block by mutation of the pore domain of the Arabidopsis inward-rectifying K+ channel KAT1.
Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K+ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K+ channel blockers are potent tools for studying the physiological functions and structural properties of K+ channels. In the present study the structural and biophysical mechanisms of Cs+ and TEA+ block of a cloned Arabidopsis inward-rectifying K+ channel (KAT1) were analyzed. Effects of the channel blockers Cs+ and TEA+ were characterized both extracellularly and intracellularly. Both external Cs+ and TEA+ block KAT1 currents. A mutant of KAT1 ("m2KAT1"; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs+ and TEA+ than the wild-type K+ channel. Internal TEA+ and Cs+ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K+ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K+ channels.
Ichida
A M
AM
Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0116, USA.
Schroeder
J I
JI
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002412:Cations / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics*; D002586:Cesium / Q000494:pharmacology; D004553:Electric Conductivity; D005110:Extracellular Space / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics*; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D002412:Cations; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D002586:Cesium; D019789:Tetraethylammonium