Klein R C, Warder S E, Galdzicki Z, Castellino F J, Prorok M
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Neuropharmacology. 2001 Dec;41(7):801-10. doi: 10.1016/s0028-3908(01)00119-8.
The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] displayed 5.6- and 8.4-fold decreases in tau(off), respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.
利用全细胞膜片钳技术对芋螺毒素 -T（con -T）、芋螺毒素 -R（con -R）及其变体进行了表征，以评估不同残基对培养的胚胎小鼠海马神经元中 N -甲基 -D -天冬氨酸（NMDA）受体抑制的起始和恢复的贡献。所得结果表明，芋螺毒素的两个最 C 端的γ-羧基谷氨酸（Gla）残基虽然对活性不是必需的，但能与受体更紧密地结合。具体而言，与亲本肽相比，con -T[γ10K/γ14K]和 con -R[γ11A/γ15A]的解离半衰期（tau(off)）分别降低了 5.6 倍和 8.4 倍。对于截短的 con -T 变体 con -T[1 - 9/Q6G]和含肌氨酸（Src）的变体 con -T[1 - 9/G1Src/Q6G]，与 con -T 相比，其解离半衰期分别快了 80 多倍和 40 多倍。对于后一种肽，共同施加 300 μM 的精胺可使起始速率常数从 3.1×10³ M⁻¹·s⁻¹提高到 12.6×10³ M⁻¹·s⁻¹。通过使用程 - 普鲁索夫方程分析平衡剂量抑制曲线，得到该肽的抑制常数（K(i)）值为 1.1 μM。相对于 NMDA，con -T[1 - 9/G1Src/Q6G]表现出明显的竞争性抑制模式。对数据进行 Schild分析得出，con -T[1 - 9/G1Src/Q6G]与受体相互作用的平衡解离常数为 2.4 μM。
