Molecular microfluorometry: Converting arbitrary fluorescence units into absolute molecular concentrations to study binding kinetics and stoichiometry in transporters

Cotransporters use energy stored in Na⁺ or H⁺ gradients to transport neurotransmitters or other substrates against their own gradient. Cotransport is rapid and efficient, and at synapses it helps terminate signaling. Cotransport in norepinephrine (NET), epinephrine (EpiT), dopamine (DAT), and serotonin (SERT) transporters couples downhill Na⁺ flux to uphill transmitter flux. NETs, for example, attenuate signaling at adrenergic synapses by efficiently clearing NE from the synaptic cleft, thus preparing the synapse for the next signal. Transport inhibition with tricyclic antidepressants prolongs neurotransmitter presence in the synaptic cleft, potentially alleviating symptoms of depression. Transport inhibition with cocaine or amphetamine, which respectively block or replace normal transport, may result in hyperactivity. Little is known about the kinetic interactions of substrates or drugs with transporters, largely because the techniques that have been successful in discovering transporter agonists and antagonists do not yield detailed kinetic information. Mechanistic data are for the most part restricted to global parameters, such as K _m and V _max, measured from large populations of transporter molecules averaged over thousands of cells. Three relatively new techniques used in transporter research are electrophysiology, amperometry, and microfluorometry. This review focuses on fluorescence-based methodologies, which - unlike any other technique - permit the simultaneous measurement of binding and transport. Microfluorometry provides unique insights into binding kinetics and transport mechanisms from a quantitative analysis of fluorescence data. Here we demonstrate how to quantify the number of bound substrate molecules, the number of transported substrate molecules, and the kinetics of substrate binding to individual transporters. Although we describe experiments on a specific neurotransmitter transporter, these methods are applicable to other membrane proteins.