Louis J. DeFelice

Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression

Antidepressant- and cocaine-sensitive serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) dictate clearance of extracellular 5-HT after release. To explore protein kinase C-mediated SERT regulation, we generated a stable human SERT (hSERT)-expressing cell line (293-hSERT) and evaluated modulation of 5-HT activity via studies of 5-HT flux, hSERT-mediated currents under voltage clamp, and surface distribution of SERT protein. 293-hSERT cells exhibit saturable, high-affinity, and antidepressant-sensitive 5-HT uptake as well as hSERT-dependent whole-cell currents. In these cells, the protein kinase C activator β-PMA caused a time-dependent reduction in 5-HT uptake capacity (Vmax) after acute application and a reduction in SERT-mediated currents. Effects of β-PMA were mimicked by the phorbol ester β-PDBu, were not observed with the inactive α-isomers, and could be blocked by treatment of cells with the protein kinase C inhibitor staurosporine. Biotinylation/immunoblot analyses showed that activity reductions are paralleled by a staurosporine-sensitive loss of surface SERT protein. These data indicate that altered surface abundance, rather than reduced catalytic transport efficiency, mediates acute PKC-dependent modulation of 5-HT uptake.

Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins

Presynaptic serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) mediate antidepressant-sensitive clearance of 5-HT following release. Although we have been aware for decades that SERT-mediated 5-HT clearance can be modulated by exogenous agents including serotonin-selective reuptake inhibitors, amphetamines, and cocaine, we have had little reason to speculate that SERT activity was actively controlled through endogenous pathways. Recent studies indicate that SERTs are likely to be trafficked to specific plasma membrane subdomains to achieve localized clearance of 5-HT, and that the number of SERTs resident in the plasma membrane is controlled through kinase- and phosphatase-linked pathways. In particular, roles for protein kinase C and phosphatase 2A become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter redistribution and loss of functional uptake capacity. Based on our findings, and the studies of others in native tissues and transfected cells, we propose a model whereby SERTs are organized in a macromolecular complex in the plasma membrane that may serve to locate reuptake activity near release sites. Although many elements of this model remain hypothetical, our findings suggest a much more dynamic picture of transporter-mediated 5-HT reuptake than typically described and suggest opportunities both for the development of new SERT regulatory agents and for the identification of regulatory pathways that may be compromised in mental illness.

Relationship between membrane excitability and single channel open-close kinetics

We have developed a novel technique for simulating the influence of the effects of single channel kinetics on the voltage changes associated with membrane excitability. The technique uses probability distribution functions for the durations of channel open- and closed-state lifetimes, which can be calculated for any model of the ion conductance process. To illustrate the technique, we have used the Hodgkin and Huxley model of nerve membrane ion conductances to simulate channel kinetics during predetermined voltage changes, such as a voltage jump and an action potential. We have also simulated the influence of channels on voltage changes in a free running, non-voltage-clamped patch of membrane 1 micron2 or less in area. The latter results provide a direct illustration of the relationship between fluctuations of membrane excitability and fluctuations in channel open- and closed-state lifetimes.

Limitations of the Hodgkin-Huxley formalism: effects of single channel kinetics on transmembrane voltage dynamics

A standard membrane model, based on the continuous deterministic Hodgkin-Huxley equations, is compared to an alternative membrane model, based on discrete stochastic ion channel populations represented through Markov processes. Simulations explore the relationship between these two levels of description: the behavior predicted by the macroscopic membrane currents versus the behavior predicted by their microscopic ion channels. Discussion considers the extent to which these random events underlying neural signals mediate random events in neural computation.

Inactivation of L-type Ca channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol

This article shows that colchicine and taxol strongly influence the kinetics of L-type Ca channels in intact cardiac cells, and it suggests a mechanism for this action. It is known that colchicine disassociates microtubules into tubulin, and that taxol stabilizes microtubules. We have found that colchicine increases the probability that Ca channels are in the closed state and that taxol increases the probability they are in the open state. Moreover, taxol lengthens the mean open time of Ca channels. In this regard, taxol is similar to Bay-K 8644; however, Bay K works on inside-out patches, but taxol does not. Neither colchicine nor taxol alters the number of Ca channels in a patch. We have quantified these results as follows. It is known that L-type channels in embryonic chick heart ventricle cells have voltage- and current-dependent inactivation. In 10 mM Ba, channel conductance is linear in the range -10 to 20 mV. The conductance is 12 +/- 1 pS, and the extrapolated reversal potential is 42 +/- 2 mV (n = 3). In cell-attached patches, inactivation depends on the number of channels. One channel (holding at -80 mV and stepping to 0 mV for 500 ms) shows virtually no inactivation. However, three channels inactivate with a time constant of 360 +/- 20 ms (n = 6). In similar patches, colchicine (80 microM for 15 min) decreases the inactivation time constant to 162 +/- 33 ms (n = 4) and taxol (50 microM for 10 min) virtually abolishes inactivation (time constant 812 +/- 265 ms (n = 4)). We suggest that colchicine and taxol affect Ca channels through their action on the cytoskeleton, which in turn regulates the effective concentration of inactivating ions near the mouths of channels. An alternate explanation is that free tubulin interacts directly with Ca channels.

Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation.

Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP+ fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.

Chaotic states in a random world: Relationship between the nonlinear differential equations of excitability and the stochastic properties of ion channels

Excitable membranes allow cells to generate and propagate electrical signals. In the nervous system these signals transmit information, in muscle they trigger contraction, and in heart they regulate spontaneous beating. A central question in excitability theory concerns the relationship between the aggregate properties of membranes (marcoscopic) and the properties of channels in the membranes (mircoscopic). Hodgkin and Huxley (1952) laid the foundations of membrane excitability, and Neher and Sakmann (1976) developed techniques to study individual channels. This article focuses on the relationship between the macroscopic domain, in which non-linear differential equations determine the electrical properties of cells, and the microscopic domain, in which the probabilistic nature of channels establishes the pattern of activity. Using nerve cell membranes as an example, we examine how information in one domain predicts behavior in the other. We conclude that the probabilistic nature of channels generates virtually all macroscopic electrical properties, including resting potentials, action potentials, spontaneous firing, and chaos.

Ionic interactions in the Drosophila serotonin transporter identify it as a serotonin channel.

Serotonin transporters (SERTs) are targets for drugs such as Prozac that increase serotonin (5HT) levels by blocking 5HT reuptake. Although SERTs saturate in the micromolar range, synaptic 5HT may exceed 1 mM. To examine SERTs response to high 5HT concentrations, we expressed Drosophila SERT (dSERT) in Xenopus oocytes and found that transport continued to increase with concentration up to 0.3 mM 5HT. As 5HT is a monovalent cation, its entry through an ion channel in SERT might explain uptake at high concentrations. We therefore investigated dSERT using traditional ion channel methods, including mole-fraction experiments under voltage clamp. We propose that SERTs may function as 5HT-permeable channels, and that this mechanism may be important for clearance of the neurotransmitter at high concentrations.

Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission

The Caenorhabditis elegans (C. elegans) dopamine (DA) transporter (DAT-1) regulates DA signaling through efficient DA reuptake following synaptic release. In addition to its DA transport function, DAT-1 generates detectible DA-gated currents that may influence neuronal excitability. Previously, we provided evidence that single Cl-channel events underlie DAT-1 currents. In these studies, we identified a distinct population of altered DAT-1 currents arising from DAT-1 transgenic constructs bearing an N-terminal GFP fusion. The presence of these channels suggested disruption of an endogenous regulatory mechanism that modulates occupancy of DAT-1 channel states. A leading candidate for such a regulator is the SNARE protein syntaxin 1A (Syn1A), previously found to interact with homologous transporters through N-terminal interactions. Here we establish that UNC-64 (C. elegans Syn1A homologue) associates with DAT-1 and suppresses transporter channel properties. In contrast, GFP::DAT-1 is unable to form stable transporter/UNC-64 complexes that limit channel states. Although DAT-1 and GFP::DAT-1 expressing DA neurons exhibit comparable DA uptake, GFP::DAT-1 animals exhibit swimming-induced paralysis (SWIP), a phenotype associated with excess synaptic DA release and spillover. We propose that loss of UNC-64/DAT-1 interactions leads to enhanced synaptic DA release, providing a novel mechanism for DA neuron sensitization that may be relevant to mechanisms of DA-associated disorders.

Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.

1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole‐cell recording with a voltage ramp in the range ‐90 to 50 mV revealed GABA‐induced currents (approximately ‐100 pA at ‐60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA‐induced currents were observed in parental HeLa cells or in mock‐transfected cells. 2. GABA‐induced currents were suppressed by extracellular perfusion with GABA‐free solutions or addition of GAT1 inhibitors SKF89976‐A or SKF100330‐A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis‐Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis‐Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range ‐90 to ‐30 mV. 3. GABA‐induced currents reverse direction in the range 5‐10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive‐labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1‐mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s‐1 under nominal conditions (V = ‐60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns‐2), assuming published turnover rates (approximately 10 s‐1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed‐stoichiometry transporter mode of conduction.