当前位置： SCI文献检索 > JOURNAL OF NEUROSCIENCE METHODS期刊下所有文献 > Potential misconceptions in dopamine transporter assays arising from the binding of [125I]RTI-121 to filters: effect of ions and cocaine.

Potential misconceptions in dopamine transporter assays arising from the binding of [125I]RTI-121 to filters: effect of ions and cocaine.

Abstract：

: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.

journal_name

J Neurosci Methods

journal_title

Journal of neuroscience methods

authors

Chen NH,Ding JH,Wang YL,Reith ME

doi

10.1016/s0165-0270(97)00070-8

subject

Has Abstract

pub_date

1997-08-22 00:00:00

pages

179-86

issue

2

eissn

0165-0270

issn

1872-678X

pii

S0165-0270(97)00070-8

journal_volume

75

pub_type

杂志文章

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