Preparation and Characterization of Biotinylated Analogs of the Calcium Channel Blocker ω-Conotoxin

✍ Scribed by O.T. Jones; A.P. So

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 486 KB
• Volume: 214
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1481

✦ Synopsis

We have prepared a series of biotinylated analogs of (\omega)-conotoxin ( (\omega \mathrm{CgTx}) ) as potent, selective markers for (\mathrm{N}-) type calcium channels. At (\mathbf{p H} 9.5), reaction of (\omega \mathrm{CgTx}) with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24 , or at both positions. Kinetic competition assays of ({ }^{125} \mathrm{I}) (\omega) CgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 (\times 10^{6} \mathrm{~m}^{-1} \mathrm{~s}^{-1}) ) but not dissociation (1-4 (\times) (10^{-4} \mathrm{~s}^{-1}) ). Comparison with rate constants obtained for the association ( (1.2 \times 10^{7} \mathrm{~m}^{-1} \mathrm{~s}^{-1}) ) and dissociation ( (5 \times) (10^{-8} \mathrm{~s}^{-1}) ) of native (\omega \mathrm{CgTx}) indicates that while biotinylation reduces (\omega \mathrm{CgTx}) potency (\left(K_{\boldsymbol{d}{\mathrm{kin}}}=k{-2} / \boldsymbol{k}{2}=4 \mathrm{pM}\right.) for (\omega \mathrm{CgTx})), binding of these labels to membranes is nevertheless of very high affinity ( (\boldsymbol{K}{d_{\text {kia }}} 0.1-0.3 \mathrm{nM}) ). c 1993 Academic Press, Inc.