Diffusion pathways to critical cysteines in the vesicular acetylcholine transporter of Torpedo.
Previous work had demonstrated that organomercurial-mediated modification of two cysteine residues in the vesicular acetylcholine transporter (VAChT) from Torpedo californica inhibits binding of vesamicol. The cysteines are protected by acetylcholine and vesamicol (Keller et al. 2000. J. Neurochem. 74:1739-1748). Modified "cysteine 1" is accessible to glutathione from the cytoplasmic surface, whereas modified "cysteine 2" is not. Different organomercurials and aqueous environments were used here to characterize diffusion pathway(s) leading to the cysteines. para-Chloromercuriphenylsulfonate modifies VAChT much more slowly than do more hydrophobic p-chloromercuribenzoate and phenylmercury chloride. Permeabilization of vesicles with cholate detergent increases the rate of modification by p-chloromercuriphenylsulfonate. Permeabilization does not affect the ability of glutathione to reverse modification by p-chloromercuriphenylsulfonate. Higher ionic strength causes about four-fold increase in the rate of modification. The results suggest that hydrophobic and electrostatic barriers inhibit modification of Torpedo VAChT by negatively charged organomercurials and glutathione cannot reach cysteine 2 from either side of the membrane.
Keller
James E
JE
Department of Chemistry and Biochemistry, The Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
Parsons
Stanley M
SM
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D003545:Cysteine / Q000187:drug effects*; D004058:Diffusion; D007477:Ions; D026901:Membrane Transport Proteins; D009941:Organomercury Compounds / Q000494:pharmacology*; D009994:Osmolar Concentration; D010539:Permeability; D010663:Phenylmercury Compounds / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism*; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D002352:Carrier Proteins; D007477:Ions; D026901:Membrane Transport Proteins; D009941:Organomercury Compounds; D010663:Phenylmercury Compounds; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D012965:Sodium Chloride; D020245:p-Chloromercuribenzoic Acid; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; C008859:phenylmercuric chloride