IN-VITRO ANALYSIS OF ION CHANNELS IN PERIAXOLEMMAL-MYELIN AND WHITE-MATTER CLATHRIN-COATED VESICLES - MODULATION BY CALCIUM AND GTP-GAMMA-S

This study reports the analysis of K+ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+ channels were observed with amplitudes of 25-30, 45-55, and 80-100 pS, all of which exhibited mean open-times of 1-2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+ channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+ was added to the cis side of the bilayer. Addition of 10 mu M total Ca2+ also resulted in little change in K+ channel activity. However, at 80 mu M total Ca2+ all K+ channel activity was suppressed along with the activation of a 100 pS Cl- channel. The K+ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTP gamma S to the trans side of the bilayer resulted in a restriction of activity to the 45-50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the alpha subunits of G(0), G(s alpha), and G(i alpha) and the low molecular weight G protein, ras. As with periaxolemmal-myelin treated with GTP gamma S, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1-6 msec. Addition of GTP gamma S to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K+ channel was also activated by the 6-methyl-dihydropyron-2-one (20 mu M).