Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Abstract

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation 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 neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application 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.Transporter-mediated clearance of neurotransmitters after vesicular release represents a crucial mechanism for temporal and spatial regulation of chemical signaling (1-5). Transporters for the biogenic amines dopamine, norepinephrine (NE), and serotonin (5HT) have particular significance as targets for clinically relevant psychoactive agents including, cocaine, antidepressants, and amphetamines (6). Cocaine and antidepressants are transporter antagonists that act with varying degrees of specificity to enhance synaptic concentrations of amines by limiting clearance (2, 6-8). Amphetamines enhance transporter mediated efflux in concert with a depletion of vesicular amine stores (8, 9). The localization of cocaine and antidepressant-sensitive transporters to the presynaptic neuron extends their influence to homeostatic relationships for transmitter biosynthesis and release.To date, the mechanisms of plasma membrane amine transport have been inferred primarily through transmitter-based flux measurements on populations of cells of isolated membrane fractions (10, 11). These assays predict the transport of amines to be coupled energetically to the inward movement of Na and Cl ions. In the case of 5HT transporters, this flux is coupled to the counter transport of K ions (11, 12), rendering 5HT transporters theoretically electroneutral. In the case of the NE transporter, the coupling stoichiometry predicted from cell and membrane vesicle flu...

Serotonin Transporter Function and Pharmacology Are Sensitive to Expression Level

“…Furthermore, all properties of SERT function were not sensitive to expression; for example, I Channel-like activity in heterologous expression systems is generally ascribed to GAT/NET transporters because (a) transmitter-induced currents correlate with transport and radioligand binding in transfected or injected cells and are absent from naïve controls, (b) transport and current exhibit similar ionic requirements and inhibitor sensitivities (see Refs. 20, 48, and 49 for reviews), and (c) channel events correlate with NE spikes patches containing hNET (50). For SERT we observed significant ⌽ 5-HT even under conditions when I (5-HT) was negligible but never the reverse, indicating that current was present only in cells with transport activity.…”

Amphetamine induces dopamine efflux through a dopamine transporter channel

“…To determine whether AMPH-induces DA efflux through this transporter-like pathway, we coupled patch clamp with amperometry to record hDAT ionic currents and DA efflux simultaneously (Fig. 5) (14). Briefly, a 5-m carbon fiber held at ϩ700 mV was positioned Ϸ1 m from the outer leaflet of the membrane.…”

“…To monitor the activity of DATs in isolated membrane patches from DAT-expressing cells, we used the patch-clamp technique in the outside-out configuration (13,14). In this configuration, the intracellular side of the plasma membrane faces the pipette solution, whereas the extracellular side faces the bath solutions (Fig.…”

“…Analysis of the electrical currents generated by the homologous NET (13)(14)(15), SERT (16), GAT-1 (17), and, recently, Caenorhabditis elegans DAT-1 (18) has revealed low-probability, channel-like events that correspond to a rapid, inward flux of ions and͞or substrate molecules (14). Here, we report that AMPH induces channel-like activity of the hDAT that supports the efflux of DA.…”

“…The outside-out membrane-excised patch was held Ϸ1 m from the carbon fiber, which was held at ϩ700 mV for DA oxidation (14,25,26). The carbon fiber electrode did not affect voltage clamping of the patch membrane.…”