Scaffolding proteins in highly purified rat olfactory cilia membranes.
Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.
Saavedra
María Verónica
MV
Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile.
Smalla
Karl-Heinz
KH
Thomas
Ulrich
U
Sandoval
Soledad
S
Olavarria
Karel
K
Castillo
Karen
K
Delgado
María Graciela
MG
Delgado
Ricardo
R
Gundelfinger
Eckart D
ED
Bacigalupo
Juan
J
Wyneken
Ursula
U
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002923:Cilia / Q000378:metabolism / Q000502:physiology*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism; D051528:Guanylate Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D007473:Ion Channels / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins; D051842:Olfactory Marker Protein / Q000378:metabolism*; D009831:Olfactory Mucosa / Q000378:metabolism*; D018034:Olfactory Receptor Neurons / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C518239:Cnga2 protein, rat; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D007527:Isoenzymes; D008051:Lipid Bilayers; C523118:Lzts3 protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D051842:Olfactory Marker Protein; C120532:Shank3 protein, rat; C106868:CASK kinases; D051528:Guanylate Kinases; D000262:Adenylyl Cyclases; C421784:adenylate cyclase 3