Article Figures & Data
Figures
- Figure 1.
TTX, baclofen, and GABA all inhibit inward and outward currents. Representative traces of whole-cell currents recorded from cultured rat mitral cells in the absence and presence of 0.2 µM TTX (A), 10 µM baclofen (B), or 100 µM GABA (C). The top traces show the control currents evoked from a holding potential of –70 mV in steps from –80 to 60 mV in 10-mV intervals. The middle traces show the residual currents after application of TTX (A), baclofen (B), or GABA (C). The bottom traces show TTX-sensitive (A), baclofen-sensitive (B), or GABA-sensitive (C) current that is obtained by subtraction of the residual current (after drug application) from the control current (before drug application). After application of TTX, baclofen, or GABA, the outward current reduction at 40 mV was 26.8 ± 2.4% (n = 12, p < 0.0001), 30.4 ± 2.5% (n = 12, p < 0.0001), or 22.9 ± 2.1% (n = 5, p < 0.0001), respectively. The I-V plot determined from steady-state values of drug-sensitive current is given below each group of sample traces. The removed inward component of current is indicated by an arrow. Note that the current/voltage plots below the current traces indicate steady state current measured ∼250 ms after the initiation of the step pulses. Because of the overlap of rapidly activating inward and outward currents at the initiation of the step pulses, the currents shown in the first few milliseconds of the traces may not be an accurate depiction of their kinetic properties.
- Figure 2.
The GABA-B receptor (GABAB-R1) colocalizes with sodium-activated potassium channels (SLO2.1) and voltage-sensitive sodium channels (NaV1.6). Rat olfactory bulb primary neurons were immunostained with pairwise combinations of primary antibodies (indicated at top left) raised from different species, followed by staining with fluorophore-coupled secondary antibodies. Panels c, f, and i are merged images, demonstrating that both proteins are present in the same cell. As with other channels and receptors, there is likely an intracellular pool as well a cell-surface pool, consistent with previous results (Panzanelli et al., 2004). Control samples incubated without any primary antibody show negligible staining (panels j, k, and l). The last column shows additional examples of immunostaining for SLO2.1 or NaV 1.6 in the absence or presence of the antigenic peptide (panels m, n, o, and p); the immunostaining is dramatically reduced in the presence of the peptide. Scale bar in p: 5 μm.
- Figure 3.
Similar persistent inward currents are inhibited by TTX, baclofen, and GABA. The top panels show examples of whole-cell ramp currents recorded from cultured rat mitral cells in the absence (black) and presence (red) of 20 µM quinidine. A slow ramp protocol from a holding potential of –70 mV to 10 mV in 600 ms was applied. The blue traces shown in the middle panel are the ramp currents recorded from the same cells after addition of 0.2 µM TTX (A), 10 µM baclofen (B), or 100 µM GABA (C). The traces below show the inward current components removed by TTX (A), baclofen (B), and GABA (C) obtained by subtraction of blue from red traces in the panels above. Na+ and Ca2+ were absent in the intracellular pipette solution, and Ca2+ was absent in the extracellular recording solutions. D, Averaged TTX-sensitive (blue) and baclofen-sensitive (red) ramp current traces from normalized drug-sensitive traces are similar. The averaged TTX-sensitive ramp current reaches a peak value of –181 ± 40 pA at –10.4 ± 4.3 mV (n = 6). The averaged baclofen-sensitive ramp current reaches a peak value of –193 ± 77 pA at –15.2 ± 5.1 mV (n = 6). E, F, Prior treatment with TTX does eliminate the effect of baclofen and vice versa, suggesting that both agents eliminate the same current. Representative I-V plots show that neither inward sodium current nor outward potassium current is further reduced by the other agent after exposure to one of the agents. Cultured rat mitral cells were first treated with 0.2 µM TTX or 10 µM baclofen for 10 min, then with coapplication of 0.2 µM TTX or 10 µM baclofen, respectively, for another 5 min. Application of 10 µM baclofen in the presence of 0.2 µM TTX does not significantly reduce outward current (4.3 ± 2.1%, n = 4, p > 0.05), nor does coapplication of 0.2 µM TTX in the presence of 10 µM baclofen (2.2 ± 1.0%, n = 5, p > 0.05).
- Figure 4.
Baclofen has no direct effect on SLO2.1 channels. A, Whole-cell currents of SLO2.1 channels expressed in Xenopus oocytes before (control) and after the addition of 10 μM baclofen. The currents were evoked by voltage pulses from –100 to 40 mV in 10-mV steps at a holding potential of –80 mV. B, Representative I-V plot of currents from A shows that baclofen has no direct effect on SLO2.1 currents. Plot in C summarizes the current measurements at 40 mV before (control) and after baclofen treatment. Current values were normalized by control. Mean value (black horizontal line) and SD error bar are shown. There is no significant change in current amplitude after baclofen treatment (n = 5, p > 0.05).
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