Dual-electrode voltammetry of catecholamine transport: simultaneous monitoring of uptake and efflux.
Dual-electrode voltammetry is used to characterize induced efflux of dopamine by m-tyramine via the norepinephrine transporter, stably expressed in a LLC-PK(1) cell line. A rotating disk electrode measures solely the dopamine oxidation current, while a stationary electrode held at a higher potential measures both the dopamine and m-tyramine concentrations. The ratio of the rate of dopamine efflux to the rate of m-tyramine uptake exhibits a hyperbolic dependence on initial dopamine concentration (half-maximal initial concentration of 4 microM) and is independent of the concentration of m-tyramine used to induce efflux for the two concentrations of tyramine tested (3 and 10 microM). These results are consistent with the alternating access model of transport, in which the ratio is shown to be independent of the nature of the external substrate used to induce efflux. At a representative initial dopamine concentration of 1 microM, the ratio of efflux to uptake is approximately 0.15 at the time of external substrate addition (zero internal tyramine). The result suggests that at this point, the transporter reorients to the external facing configuration without dopamine approximately 85% of the time, or expressed differently, seven external substrate molecules are taken up, on average, for each one transported outward.
Reed
Brian
B
Department of Chemistry, Emory University, Atlanta, GA, USA. reedb@rockefeller.edu
Chen
Nianhang
N
Justice
Joseph B
JB
D000818:Animals; D001692:Biological Transport; D004298:Dopamine / Q000493:pharmacokinetics*; D004566:Electrodes; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D018374:LLC-PK1 Cells; D050484:Norepinephrine Plasma Membrane Transport Proteins; D013552:Swine; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000493:pharmacokinetics
D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D014443:Tyrosine; C015566:3-tyrosine; D004298:Dopamine