This project concerns the transport of ion channels in nerve axoplasm by intracellular organelles. Previous work in this project has demonstrated to distinct populations of organelles isolated from the axoplasm of squid giant axons; a putative anterograde organelle population having diameters in the 40-60 nm range, and a putative retrograde population having diameters in the 100-150 nm range. During the past year these two populations have been analyzed with electrophysiological and biochemical procedures. One key finding has been that immunoblots of the anterograde organelles display cross-reactivity to an antibody against kinesin, a 120 kD ATPase which translocates these organelles along microtubules in the nerve. No cross-reactivity to kinesin was observed in the putative retrograde organelles even though the antibody has been shown with differential interference contrast light microscopy to block transport of organelles in both the anterograde and retrograde directions. Preliminary work has also been carried out with antibodies raised against various portions of the C-terminal region of the rat brain potassium channel, Kv 2.1. One particular antibody shows specific crossreactivity in immunoblots to the retrograde organelles. However, the antibody does not block potassium current, l/K, in axons using the standard axial wire voltage-clamp technique, whereas other antibodies which do block l/K in the axon do not appear to exhibit cross-reactivity with either population of organelles in immunoblots. These results suggest that the l/K channel has biochemical differences depending upon its location, be it either in anterograde or retrograde organelles, or in the axolemma itself.
