Localization of voltage-gated K+ channels in squid giant axons

We have localized the classical voltage-gated K+ channel within squid giant axons by immunocytochemistry using the Kv1 antibody of Rosenthal et al, (1 996), Widely dispersed patches of intense immunofluorescence were observed in the axonal membrane. Punctate immunofluorescence was also observed in th e axoplasm and was localized to similar to 25-50-mum-wide column down the l ength of the nerve (axon diameter similar to 500 mum). Immunoelectronmicros copy of the axoplasm revealed a K+ channel containing vesicles, 30-50 nm in diameter, within this column, These and other vesicles of similar size wer e isolated from axoplasm using a novel combination of high-speed ultracentr ifugation and controlled-pore size, glass bead separation column techniques . Approximately 1% of all isolated vesicles were labeled by K+ channel immu nogold reacted antibody. Incorporation of isolated vesicle fractions within an artificial lipid bilayer revealed K+ channel electrical activity simila r to that recorded directly from the axonal membrane by Llano ct al, (1988) , These K+ channel-containing vesicles may be involved in cycling of K+ cha nnel protein into the axonal membrane, We have also isolated an axoplasmic fraction containing similar to 150-nm-diameter vesicles that may transport K+ channels back to the cell body. (C) 2000 John Wiley & Sons, Inc.