N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) is a selective persistent inhibitor of glycine transport

Abstract

1. The regulation of glycine concentrations within excitatory synapses is poorly understood and it has been proposed that the GLYT1 subtypes of glycine transporters play a critical role in determining resting concentrations of glycine. Selective GLYT1 inhibitors may provide pharmacological tools to probe the dynamics of synaptic glycine concentrations, which may influence the activation properties of NMDA receptor activity. 2. We have characterized the selectivity and mechanism of action of the glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS). The glycine transporters, GLYT1a, b and c and GLYT2a were expressed in Xenopus laevis oocytes and two electrode voltage clamp techniques and radiolabelled (3)H-glycine flux measurements were used to characterize the effects of NFPS on glycine transport. 3. NFPS inhibits glycine transport by the GLYT1a, b and c subtypes of glycine transporters, but has no effect on the GLYT2a subtype of transporter. We show that NFPS does not attain its specificity via an interaction with the Na(+), Cl(-) or glycine site, nor does it act at an intracellular site. NFPS inhibition of glycine transport is time and concentration dependent and inhibition of transport by NFPS persists after washout of NFPS from the bath solution, which suggests that inhibition by NFPS is long lasting.

Figure 1

The structure of NFPS. Sarcosine is the N-methylated derivative of glycine and is a competitive substrate inhibitor of GLYT1 but not GLYT2 transporters. NFPS consists of a large lipophilic tail with a sarcosine head group.

Figure 2

NFPS is a selective blocker of GLYT1 subtypes of glycine transporters. Representative traces from oocytes expressing GLYT1b (a) and GLYT2a (b) voltage clamped at −60 mV. One hundred μM glycine (solid bar) was applied first and after washout, 100 μM glycine (solid bar) and 300 nM NFPS (open bar) were co-applied for 3 min. After a further 10 min of superfusion in drug-free buffer, 300 nM NFPS alone (open bar) was applied. In the trace for an oocyte expressing GLYT2a (b) 30 μM glycine with 300 nM NFPS was applied together after a control dose of glycine (30 μM). In uninjected oocytes, application of glycine and NFPS, together or alone, did not generate a current. (c) NFPS similarly inhibited the other GLYT1 transporter subtypes. Thirty μM glycine and 300 nM NFPS were applied to oocytes expressing GLYT1a, b and c and GLYT2a as described above and the per cent inhibition measured after 3 min exposure to NFPS. Statistical analysis was carried out using the Kruskal-Wallis test. Significance (P⩽0.05) is indicated by *.

Figure 3

NFPS binding to GLYT1b does not require glycine, Na⁺ or Cl⁻. Current trace from representative oocytes expressing GLYT1b voltage clamped at −60 mV (a). In normal frog ringers buffer, 30 μM glycine was first applied (solid bar) followed by wash out in frog ringers. Three hundred nM NFPS was then applied alone for 3 min (open bar) followed by a washout period in drug-free frog ringers. Thirty μM glycine (solid bar) was then re-applied. (b) Experiments were carried out in choline substituted Na⁺ free buffer or gluconate substituted Cl⁻ free buffer. After application of 30 μM glycine to measure control responses, the bath solution was switched and allowed to equilibrate for 2 min before 300, 100 or 30 nM NFPS was applied for 3 min. After 1 min wash out of NFPS in the ion substituted buffer, the buffer was switched back to standard frog ringers buffer and 30 μM glycine reapplied. The per cent inhibition of glycine transport currents after the three NFPS treatments are presented. The absence of Na⁺ or Cl⁻ did not significantly change the level of inhibition for any of the treatments (Kruskal-Wallis test).

Figure 4

NFPS inhibits ³H-glycine uptake in oocytes expressing GLYT1b. ³H-glycine uptake by uninjected oocytes (C1, 2 and 3) and oocytes expressing GLYT1b (T1, 2 and 3) was measured as described in Methods. C1 and T1: 30 μM ³H-glycine alone. C2 and T2: 30 μM ³H-glycine and 1 μM NFPS. C3 and T3 30 μM ³H-glycine and 1 μM NFPS after 10 min pre-incubation with 1 μM NFPS. In each case the results are the mean±s.e.mean from five oocytes and Kruskal-Wallis followed by Dunns test was used to assess statistical significance. P⩽0.05 is indicated by *.

Figure 5

NFPS does not compete with glycine for the glycine binding site. A control current induced by 30 μM glycine was measured in oocytes expressing GLYT1b, voltage clamped at −60 mV. Three hundred or 30 nM NFPS was co-applied with 0, 30 or 3000 μM glycine for 3 min followed by a 5 min washout. Thirty μM glycine was applied again and compared to control currents. The extent of inhibition by 300 (a) or 30 nM (b) NFPS after 3 min application was not significantly altered in the presence of 0, 30 or 3000 μM glycine (Kruskal-Wallis test).

Figure 6

NFPS is an apparent irreversible inhibitor. (a) Representative current trace from an oocyte expressing GLYT1b voltage clamped at −60 mV. After a control (10 μM) glycine response, glycine (10 μM) was reapplied until the current reached a maximal response (point 1) followed by the co-application of 300 nM NFPS. Three minutes later NFPS was washed out of the bath, but in the continued presence of 10 μM glycine (point 2). After washout of glycine for a further 5 min, 10 μM glycine was reapplied (point 3). Currents induced by subsequent application of 10 μM glycine up to 90 min later remained equally and significantly inhibited (Kruskal-Wallis test). (b) Current responses to application of 10 μM glycine were measured at the indicated points (1 – 3) and represented in a bar graph (n=6). Significance (P⩽0.05) is indicated by *.

Figure 7

NFPS interacts with the oocyte membrane. (a) Oocytes expressing GLYT1b were exposed to 1 μM NFPS for 10 min and then washed at least three times daily for the next 3 days. Glycine transport currents were measured from oocytes 1, 2 and 3 days after NFPS exposure and compared to unexposed oocytes (Student's t-test). Currents were significantly inhibited on days 1 and 2, however on day 3, variability was high and although reduced this decrease did not reach significance. Data represents mean±s.e.mean, n⩾3 for each condition. (b) Immediately after injection of GLYT1b cRNA, NFPS was applied to the oocytes at the indicated doses for 10 min followed by extensive washing in standard buffer. Glycine transport currents were measured 3 days later and compared to unexposed oocytes. Data is mean±s.e.mean, n=3 for each condition. (c) A similar protocol to Figure 7b was used, except that 1 μM NFPS was applied for 10 min and 30 min immediately after cRNA injection. Data presented are from mean±s.e.mean, n=10 for each condition. Control experiments were also included where GLYT2a cRNA was injected instead of GLYT1b and in each case NFPS had no effect on the glycine transport currents. Data analysis for b and c was carried out using the Kruskal-Wallis test and Dunns test. Significance (P⩽0.05) is indicated by *.

Figure 8

NFPS does not inhibit transport when injected into the cytoplasm of the oocyte. A representative current trace from an oocyte expressing GLYT1b voltage clamped at −60 mV. After insertion of the voltage clamp electrodes into the oocyte, a microinjection needle, containing 20 μM NFPS, was inserted into the oocyte. A control current response to 30 μM glycine was measured (solid bar) and then 50 nL of 20 μM NFPS was injected into the oocyte (indicated by the arrow). After stabilization of the baseline current, 30 μM glycine was re-applied (solid bar) and the current measured. After washout of glycine, 30 μM glycine (solid bar) and 1 μM NFPS (open bar) were co-applied to the bath solution.

Figure 9

NFPS inhibits GLYT1b in a time and dose dependent manner. In oocytes voltage clamped at −60 mV a control glycine (30 μM) current was first established. Then NFPS, at doses from 10 to 1000 nM, was co-applied with 30 μM glycine. Inhibition was allowed to persist until currents were reduced to half the control glycine current and measured as time taken to reach half of complete inhibition (t_1/2). Data represents mean±s.e.mean with n=5 for each NFPS dose.