K(+)-channel transgenes reduce K(+) currents in Paramecium, probably by a post-translational mechanism.
PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.
Ling
K Y
KY
Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA.
Haynes
W J
WJ
Oesterle
L
L
Kung
C
C
Preston
R R
RR
Saimi
Y
Y
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004594:Electrophysiology; D016368:Frameshift Mutation; D020868:Gene Silencing; D008957:Models, Genetic; D016366:Open Reading Frames; D010247:Paramecium / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D019076:Transgenes
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D015221:Potassium Channels; D004247:DNA; D002118:Calcium