The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity.
The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.
Gincel
Dan
D
Department of Life Sciences and the Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Shoshan-Barmatz
Varda
V
D000818:Animals; D001921:Brain / Q000378:metabolism; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002794:Choline / Q000494:pharmacology; D020552:Comet Assay / Q000379:methods; D005942:Gluconates / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008567:Membranes, Artificial; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D012413:Rubidium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism; D016708:Synaptophysin / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D013574:Synaptosomes / Q000378:metabolism
D002712:Chlorides; D005942:Gluconates; D007473:Ion Channels; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D016708:Synaptophysin; D002586:Cesium; D012965:Sodium Chloride; D011189:Potassium Chloride; C028019:cesium chloride; D012413:Rubidium; C032710:rubidium chloride; D002794:Choline