Fig 1.
The Amino acid sequence and disulfide bonds of TPN-RQ are illustrated. Each amino acid was classified into five groups and shown in different colors in order of reduction in ROMK blocking activity when each amino acid was changed to Ala (red >80%; orange, 60–80%; yellow, 40–60%; green, 20–40%; blue, <20% at 1 μM) (see text and Table 1).
Table 1.
ROMK blocking activities of partial and full length TPN peptides.
Fig 2.
Blockade of ROMK currents by TPN-RQ and the lack of effect of Cys-to-Ser mutants.
(A) Whole-cell ROMK currents were evoked by step-pulses, as shown in the protocol. The application of TPN-RQ decreased ROMK currents in a concentration-dependent manner. The remaining current was resistant to 6 mM BaCl2, which is a selective blocker of the ROMK channel, indicating that TPN-RQ blocked the channel nearly completely. (B and C) The C3S and C5S mutants of TPN-RQ hardly inhibited the ROMK currents even at the highest concentrations, indicating the essential role of these Cys residues. (D) I-V relationship of whole-cell ROMK channel currents in the absence (Control) or presence of blockers (7 μM TPN-RQ and 6 mM BaCl2). The I-V curves of TPN-RQ and BaCl2 were completely overlapped. (E) The concentration–response curve of TPN-RQ, (C3S), and (C5S) on ROMK conductance (n = 3). (F) Preceding treatment of TPN-RQ with PDI did not affect the blocking activity of TPN-RQ, suggesting the adequate formation of disulfide bonds (n = 4).
Fig 3.
Lack of effect of TPN-RQ on the Kir2.1 channel.
Whole-cell currents were recorded from 56–3 cells, and Kir2.1 channel currents were evoked by the step pulses. TPN-RQ hardly inhibited the 0.3 mM BaCl2-sensitive Kir2.1 currents.
Fig 4.
Chemically synthesized TPN-RQ (100 μL, 16.5 μM) was fractionated on a cation exchange column, monitoring A230 nm (A). A peak was found with a retention time of 17.5 min on the declining baseline (arrow). Then, the peptides, which were mixed with mouse serum, were analyzed (B and C). The incubation with serum did not affect the peak height or retention time. The ordinate indicates the A230 nm (arbitrary unit of HPLC UV-detector) and NaCl concentration (mM).
Fig 5.
Antidepressive effect of TPN-RQ.
(A) TPN-RQ decreased immobile time at the TST, whereas TPN-RQ(C3S) decreased the time insignificantly. (B) Dose-dependency of TPN-RQ on immobile time in the TST. (C) TPN-RQ, but not TPN-RQ(C3S), decreased the immobile time at FST. (D) Significant decrease in immobile time in the TST of the chronically stressed mice. (E) TPN-RQ insignificantly increased the latency for beginning eating in the NSF test. (F) Impairment of motor coordination by TPN-RQ. The administration of TPN-RQ significantly increased the time required to cross the central 60 cm of the beam in consecutive 4 trials (10 mm beam, 2 trials; 6 mm, 2 trials). (G) TPN-RQ decreased spontaneous rotations in the activity wheel in 4 min.
Fig 6.
TPN-RQ, but not TPN-RQ(C5S), showed an anxiogenic effect in the OFT (A), EPM (B), and LDT (C). The anxiogenic effect was dose-dependent in the EPM (D).
Fig 7.
The ROMK-selective blocker TPN-LQ showed similar behavioral effects.
(A) TPN-RQ blocked ROMK, GIRK1/2, and GIRK1/4 channels nearly equally. (B) TPN-LQ blocked ROMK channel selectively over GIRK1/2 and GIRK1/4 channels. TPN-LQ decreased immobile time in the TST (C) and the FST (D) at the same dose as TPN-RQ. TPN-LQ showed the anxiogenic effect in EPM (E) and OFT (F). TPN-LQ decreased spontaneous rotations in the activity wheel, indicating the impairment of locomotor activity (G). Another ROMK selective blocker, VU591, also showed antidepressive effect in the TST (H).
Fig 8.
Anti-c-Fos immunoreactive cell numbers in depression-, anxiety-, and stress-related nuclei.
A significant difference in the number of anti-c-Fos immunoreactive neurons was observed only in the lateral septum. (LS, lateral septum; PFC, prefrontal cortex (cingulate cortex); DG, dentate gyrus; PVN, hypothalamic paraventricular nucleus; PVT, thalamic paraventricular nucleus; LHb, lateral habenular nucleus; [ipsi, injection side; contra, opposite side] CeA, central amygdala; BLA, basolateral amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus: LV, lateral ventricle; L1, layer 1; GCL, granule cell layer; 3V, third ventricle; Aq, central aqueduct) (bar = 100 μm, n = 7 and 6).
Fig 9.
ROMK channel blocking activities of TPN mutants.
Ordinate indicates the normalized whole-cell conductances of ROMK channels treated with the indicated peptide at a concentration of 1 μM, which were estimated according to the fitted curve (n = 3–6). When the mutation resulted in the loss of the blocking activity, i.e. the remaining conductance was high, the mutated amino acid was colored red in Fig 1.