Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes

Abstract

Altered serotonin (5-HT) signaling is implicated in several neuropsychiatric disorders, including depression, anxiety, obsessive-compulsive disorder, and autism. The 5-HT transporter (SERT) modulates 5-HT neurotransmission strength and duration. This is the first study using rotating disk electrode voltammetry (RDEV) to measure 5-HT clearance. SERT kinetics were measured in whole brain synaptosomes. Uptake kinetics of exogenous 5-HT were measured using glassy carbon electrodes rotated in 500 μL glass chambers containing synaptosomes from SERT-knockout (-/-), heterozygous (+/-), or wild-type (+/+) mice. RDEV detected 5-HT concentrations of 5nM and higher. Initial velocities were kinetically resolved with K(m) and V(max) values of 99±35 standard error of regression (SER) nM and 181±11 SER fmol/(s×mg protein), respectively in wild-type synaptosomes. The method enables control over drug and chemical concentrations, facilitating interpretation of results. Results are compared in detail to other techniques used to measure SERT kinetics, including tritium labeled assays, chronoamperometry, and fast scan cyclic voltammetry. RDEV exhibits decreased 5-HT detection limits, decreased vulnerability to 5-HT oxidation products that reduce electrode sensitivity, and also overcomes diffusion limitations via forced convection by providing a continuous, kinetically resolved signal. Finally, RDEV distinguishes functional differences between genotypes, notably, between wild-type and heterozygous mice, an experimental problem with other experimental approaches.

Figure 1

Schematic diagram showing rotating disk electrode voltammetry apparatus. A potentiostat applies a 550 mV potential to a working electrode lowered into a custom glass chamber and rotated at 3000 RPM. Synaptosomes are kept at physiological temperature (37° C) by a water recirculator (solid-tip arrows). The analog signal is converted to digital and recorded on a computer. The inset shows the convective pathways of laminar solution flow (line-tip arrows) caused by rotation of the working electrode.

Figure 2

Hydrodynamic voltammagram, Nernst plot, and oxidation scheme of 5-HT in physiological buffer. (A) A hydrodynamic voltammagram of 1 μM 5-HT in pH 7.4 physiological buffer and background (buffer only). Each datum point is the mean ± SEM of three separate determinations. Error bars for open circles fall within the dimensions of the symbol. (B) Expanded dotted box from Panel A shows the background-subtracted plot. (C) Nernst plot of points shown in Panel B. (D) Scheme adapted from Wrona and Dryhurst, 1990.shows that complete oxidation of serotonin liberates two electrons and one proton, while incomplete electrolysis frees a proton and an electron, producing a radical intermediate.

Figure 3

Linear electrode responses and sample oxidation current showing noise. (A) The electrode had a linear response to a physiologically relevant range of 5-HT (50 nM to 1 μM) in pH 7.4 buffer (n = 10 - 18 per concentration). The presence of tissue (synaptosomes) (n = 7 - 8 per concentration) with or without 1 μM paroxetine (n = 5 - 9 per concentration) did not affect the magnitude of the electrode's response to 5-HT. All three lines are coincident. Nearly all error bars fall within the dimensions of the symbols, and the symbols superimpose. (B) Raw data trace showing ratio of signal to noise and change in current when adding 50 nM 5-HT (arrow) to buffer. The boxed region is enlarged in the inset to show the magnitude of the noise. RMS noise was 0.33 ± 0.03 nA (n=5). This value was used with the coincident linear plots in (A) to calculate a detection limit of 4.8 ± 0.4 nM. Data are expressed as mean ± SEM.

Figure 4

Measuring 5-HT uptake with rotating disk electrode voltammetry. (A) A time course shows an entire recording of an oxidation current, from the time the potential is applied to the time that the 5-HT is completely taken up by the synaptosomal tissue. Achieving a flat baseline takes approximately 12 - 15 minutes, during which non-specific components in the preparation are oxidized by the electrode. Panel B shows expansion of dotted box in (A). (B) Initial rates are obtained from the linear slope (dotted line) of a tangent line to the steepest portion of the uptake curve, which represents the initial apparent zero order portion of the plot. Change in current/time is converted to fmol 5-HT taken up/s and normalized to protein concentrations (mg).

Figure 5

Kinetic analysis of SERT and SERT function in different SERT genotypes. (A) Raw data traces demonstrating inhibition of clearance by paroxetine in SERT +/+ synaptosomes. Background drift has been subtracted from the traces. (B) Michaelis-Menten plots showing how SERT activity in SERT +/+ synaptosomes (star symbols) is determined by subtracting initial rates in the presence of 1 μM paroxetine (open circles) from control initial rates (vehicle, closed circles). Drift was not subtracted from these data. The K_m for SERT was 99 ± 35 nM and V_max 182 ± 11 fmol / (s × mg). (C) RDEV detects differences in SERT function in synaptosomes isolated from SERT +/+, +/−, and −/− mice in taking up 100 nM 5-HT, a concentration near SERT's calculated K_m. The initial velocity of SERT was 88.2 ± 10.2 fmol / (s × mg) in +/+ synaptosomes (n = 5), 47.8 ± 11.2 fmol / (s × mg) in +/− synaptosomes (n=8), and −5.8 ± 16.7 fmol / (s × mg) in −/− synaptosomes (n = 5). SERT velocity significantly differed between all pairs of genotypes: wild-type versus het, *p<0.05, +/− versus −/−, **p<0.01, wild-type versus knockout, ***p<0.001. Data are expressed as mean ± SEM. Data were analyzed by a one-way ANOVA with post hoc Newman-Keuls multiple comparisons tests comparing each pair of genotypes. The significance level was set at p<0.05.