Membrane potential manipulation in synaptic plasma membrane vesicles for studying neurotransmitter uptake and release.
Synaptic plasma membrane (SPM) vesicles represent a membrane fraction very useful in studying non-vesicular neurotransmitter release. The procedure described here to isolate SPM vesicles from a crude synaptosomal fraction of sheep brain cortex is quick, simple (ultracentrifugation in a discontinuous density gradient of dextran T110), and combines a high yield (130 micrograms/g brain) with a satisfactory grade of purification. The preparation of SPM vesicles consists of vesicles (approximately 0.54 +/- 0.8 micron diameter) delimited by a single membrane with the native orientation. We are able to ascertain these characteristics on the basis of morphology studies (electron microscopy observations), enzyme activities (Na+/K(+)-ATPase, Ca2+/Mg(2+)-ATPase, acetylcholinesterase and glucose-6-phosphatase), biochemical composition (lipid and protein analysis) and the tetrodotoxin sensitivity of the veratridine-induced gamma-aminobutyric acid (GABA) release. Isolating the SPM vesicles by the proposed procedure permits manipulating the ionic gradients across the membrane by changing the ion concentrations on either side or by utilizing specific ionophores. The vesicles retain their various activities, including their capacity for neurotransmitter uptake and release assays for at least 3 months, when preserved at -70 degrees C. Furthermore, the vesicles permit depicting the electrochemical gradients across the membranes into chemical and electrical components. We describe the use of the tetraphenylphosphonium cation (TPP+) to dissipate the membrane potential (delta psi) of the vesicles, while preserving ionic gradients. The characteristics of the lipid-soluble cation TPP+ allows a massive inflow of this cation into vesicular compartments and a consequent depolarization.
Gonçalves
P P
PP
Departamento de Biologia, Universidade de Aveiro, Portugal.
Carvalho
A P
AP
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D002412:Cations / Q000494:pharmacology; D004573:Electrolytes / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods*; D007202:Indicators and Reagents / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D009488:Neurosciences / Q000379:methods*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D012756:Sheep; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002412:Cations; D004573:Electrolytes; D007202:Indicators and Reagents; D018377:Neurotransmitter Agents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D014634:Valinomycin; D013779:Tetrodotoxin; D005680:gamma-Aminobutyric Acid; C013289:tetraphenylphosphonium