PATCH-CLAMP AND AMPEROMETRIC RECORDINGS FROM NOREPINEPHRINE TRANSPORTERS - CHANNEL ACTIVITY AND VOLTAGE-DEPENDENT UPTAKE

Transporters for the biogenic amines dopamine, norepinephrine, epineph rine and serotonin are largely responsible for transmitter inactivatio n after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants , cocaine, and amphetamines. Despite their prominent role in neurotran smitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single tran sporter level. The resolution of radiotracer-based flux techniques lim its the opportunities to dissect these problems, Here we combine patch -clamp recording techniques with micro-amperometry to record the trans porter-mediated flux of norepinephrine across isolated membrane patche s. These data reveal voltage-dependent norepinephrine flux that correl ates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked wit h bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmit ter across the membrane in discrete shots containing hundreds of molec ules, Amperometry is used widely to study neurotransmitter distributio n and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present applic ation is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.