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首页> 外文期刊>Journal of Neuroscience Methods >Potential misconceptions in dopamine transporter assays arising from the binding of (125I)RTI-121 to filters: effect of ions and cocaine.
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Potential misconceptions in dopamine transporter assays arising from the binding of (125I)RTI-121 to filters: effect of ions and cocaine.

机译：（125I）RTI-121与过滤器的结合引起的多巴胺转运蛋白测定中的潜在误解：离子和可卡因的作用。

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摘要

Binding of the cocaine analog 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121) to filters was studied in order to assess its contribution to labeling dopamine transporters on rat striatal synaptosomal membranes in filtration assays. Filter binding (FB) decreased with increasing Na+. Cocaine (30 and 100 microM) substantially reduced the FB at low Na+ with much less of an effect at higher Na+. Similar results were observed with K+. At 10 mM Na+, RTI-121 (1 microM) displaced the FB to the same degree as cocaine (100 microM); mazindol (10 microM), BTCP (1 microM), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 microM) did not. If the specific binding was calculated without deducting the FB displaced with cocaine (DFB), the DFB accounted for 15-19% of the 'specific binding' at 10 mM Na+ in the assay. This additional binding population resulted in an upward curvilinear Scatchard plot and incorrect estimation of equilibrium binding parameters and ion potencies. At 10 mM Na+, without deduction of DFB, the high-affinity component had a Kd of 3.4 nM and Bmax of 2.4 pmol/mg protein, and the respective values for the low-affinity component were 84 nM and 16 pmol/mg protein; when DFB was deducted, one component was observed with a Kd of 4.4 nM and Bmax of 3.3 pmol/mg protein. The presence of higher Na+ in the assay diminished these artifacts. Thus, at 150 mM Na+, without deduction of DFB, there was one binding component with a Kd of 3.9 nM and Bmax of 4.6 pmol/mg protein; these values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted.
机译：研究了可卡因类似物3β-（4- [125I]碘苯基）托烷-2β-羧酸异丙酯（[125I] RTI-121）与过滤器的结合，以评估其对标记大鼠纹状体多巴胺转运蛋白的作用。滤膜中的突触体膜。过滤器结合（FB）随Na +增加而降低。可卡因（30和100 microM）在低Na +下显着降低了FB，而在高Na +下的作用要小得多。使用K +观察到相似的结果。 Na +浓度为10 mM时，RTI-121（1 microM）将FB置换为与可卡因（100 microM）相同的程度。 mazindol（10 microM），BTCP（1 microM）和多巴胺（1 mM）程度较小;而GBR12935（1 microM）则没有。如果在不扣除用可卡因（DFB）取代的FB的情况下计算特异性结合，则在测定中，DFB占10 mM Na +下“特异性结合”的15-19％。这种额外的结合种群导致曲线Scatchard曲线向上，对平衡结合参数和离子强度的估计不正确。在10 mM Na +下，不扣除DFB，高亲和力组分的Kd为3.4 nM，Bmax为2.4 pmol / mg蛋白质，低亲和力组分的各自值为84 nM和16 pmol / mg蛋白质;当扣除DFB时，观察到一种成分，其Kd为4.4 nM，Bmax为3.3 pmol / mg蛋白。测定中较高的Na +的存在减少了这些伪影。因此，在150 mM Na +浓度下，在不扣除DFB的情况下，存在一种结合成分，其Kd为3.9 nM，Bmax为4.6 pmol / mg蛋白质。当扣除DFB时，这些值变为3.3 nM和3.8 pmol / mg蛋白质。

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