The classical voltage-gated potassium ion channel in squid giant axons has been localized using an affinity purified polyclonal antibody. Widely dispersed spots of intense immunofluorescence were observed throughout the axonal membrane: ~1 per 25 square microns of membrane surface area. We also observed punctate immunofluorescence in the axoplasm which was localized to a ~25 micron wide column down the length of the nerve (axon diameter ~500 microns). Immuno-electron microscopy revealed potassium ion channel containing transport vesicles ~20-30 nm in diameter in linear arrays within this column. Transport vesicles were isolated from axoplasm using novel techniques described in previous annual reports. Approximately 1% of all such vesicles contained a potassium ion channel. These preparations lacked synaptobrevin, the classical v-snare of synaptic vesicles. Synaptobrevin was observed in another axoplasm fraction. Incorporation of transport vesicles into artificial lipid bilayers revealed potassium ion channel activity similar to that recorded directly from the axonal membrane. Transport vesicles may be involved in recycling of axonal proteins (potassium ion channels and other proteins) via constituitive fusion. We also have isolated another axoplasm fraction containing larger vesicles (d ~ 150 nm) - possibly endocytotic in origin - which may take potassium ion channels back to the cell body. - Potassium ion channel, nerve axons, protein targeting, constituitive fusion

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Intramural Research (Z01)
Project #
1Z01NS002608-16
Application #
6290628
Study Section
Special Emphasis Panel (LNP)
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Lake, Robert J; Grimm, Lisa M; Veraksa, Alexey et al. (2009) In vivo analysis of the Notch receptor S1 cleavage. PLoS One 4:e6728
Paydarfar, David; Forger, Daniel B; Clay, John R (2006) Noisy inputs and the induction of on-off switching behavior in a neuronal pacemaker. J Neurophysiol 96:3338-48
Clay, John R (2003) On the persistent sodium current in squid giant axons. J Neurophysiol 89:640-4
Clay, John R; Kuzirian, Alan (2002) Trafficking of axonal K+ channels: potential role of Hsc70. J Neurosci Res 67:745-52
Clay, J R; Shrier, A (2002) Temperature dependence of bistability in squid giant axons with alkaline intracellular pH. J Membr Biol 187:213-23
Clay, J R; Kuzirian, A M (2001) A novel, kinesin-rich preparation derived from squid giant axons. Biol Bull 201:243-5
Clay, J R; Shrier, A (2001) Action potentials occur spontaneously in squid giant axons with moderately alkaline intracellular pH. Biol Bull 201:186-92
Clay, J R (2000) Determining K+ channel activation curves from K+ channel currents. Eur Biophys J 29:555-7
Clay, J R; Kuzirian, A M (2000) Localization of voltage-gated K(+) channels in squid giant axons. J Neurobiol 45:172-84