Detection and localization of a putative cyclic-GMP-activated channel protein in the protozoan ciliate Stentor coeruleus.
Immunoblotting and immunocytochemical assays were employed to identify and localize a channel protein activated by cyclic GMP (cGMP) in the protozoan ciliate Stentor coeruleus. Analysis of whole-cell homogenate with antibodies raised against the alpha-subunit of the cGMP-activated channel protein from bovine rod outer segments and against cGMP revealed four major protein bands with molecular masses of 40 kDa, 63 kDa, and over 120 kDa, which bound cGMP. However, only a cGMP-binding protein of 63 kDa, corresponding to the alpha-subunit of the cGMP-activated ion channel protein from bovine rod outer segments, was found in the ciliate cortex fraction. The functional cGMP-activated channel protein was also shown to be present in the cortex fraction of S. coeruleus by patch-clamp measurements of artificial liposomes. Incorporation of the cortex fraction into liposomes resulted in the appearance of ion channel activity related to cGMP. The reconstituted protein channels were strongly inhibited by l-cis-diltiazem, a known potent blocker of many types of cyclic-nucleotide-activated channels. The results presented here are the first demonstration of the existence and localization of a putative cGMP-activated channel protein in the ciliate S. coeruleus. Cyclic-nucleotide-activated channel proteins are nonspecific cation channels which mediate the receptor potentials in photoreceptor cells and in cells of the olfactory epithelium. On the basis of these data, we suggest that the 63 kDa protein identified in Stentor coeruleus is also a cGMP-activated ion channel and that it may be involved as an effector in the photosensory transduction pathway leading to the motile photophobic response in this ciliate protist.
Walerczyk
M
M
Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Fabczak
H
H
Fabczak
S
S
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D016798:Ciliophora / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008081:Liposomes; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007473:Ion Channels; D008081:Liposomes; D015800:Protozoan Proteins; C022994:cyclic GMP-binding protein; D000242:Cyclic AMP; D004110:Diltiazem; D006152:Cyclic GMP