Article Figures & Data
Figures
- Figure 1.
Protocol used for estimation of relative Cl– conductance at rest. A, Voltage protocol (below in black/gray; ramp sequences appearing as blocks; for detailed ramps, see B) and recorded current (top, red), with glycine application as indicated by the green bars (short bars, 100 µM; long bar, 1.0 mM). B, Voltage protocol (bottom) and recorded current (top) for part of the ramp train shown within dashed box in A.
- Figure 2.
[Cl–]i loading and estimation of transporter-dependent recovery capacity. A, Voltage protocol (bottom) and recorded current (top) during loading a cell with Cl–. Note that the baseline voltage during loading was –14 mV to obtain a large driving force for Cl– current evoked by 1.0 mM GABA. Data from numbered ramps are shown in B and C. B, I-V relations corresponding to the ramps (no 1-4, 6, 8, as marked in A) applied during Cl– loading (corrected for series resistance and with leak components subtracted). C, Time course of [Cl–]i during Cl– loading, as computed from ECl obtained from the I-V relations in B, and superimposed fitted exponential curve. Resting [Cl–]i was obtained from a separate, preceding ramp (not shown). D, Voltage ramps (insets), leak and GABA-evoked currents used to probe [Cl–]i during recovery after the end of Cl– loading. Ramps for [Cl–]i estimates at two different times are shown in Da and Db (compare I-V relations in E). E, I-V relations from probe currents as in D (with the relations corresponding to currents in Da and Db indicated). Note that I-V relations are shifted with time to more negative voltages, as a consequence of the changing [Cl–]i and ECl. F, Full-time course of [Cl–]i changes during loading (first 25 s) and recovery phases with superimposed mono-exponential fit (red thick line) of the recovery phase.
- Figure 3.
Pharmacology of transporter-mediated recovery of [Cl–]i. A–C, [Cl–]i recovery under control conditions (black filled circles) and in the presence of 10 µM bumetanide (A, red open circles), 0.5 mM furosemide (B, blue open circles), and 10 µM VU0255011-1 (C, orange open circles), with control and drug application for the same cell. Cl– was loaded and [Cl–]i quantified as in Figure 2. D, Summary of drug effects on the level of [Cl–]i after 10 min recovery, measured as [Cl–]i normalized to maximal [Cl–]i during Cl– loading. Note the lack of significant effect of the NKCC1-blocker bumetanide but highly significant effects of the KCC2 blockers furosemide and VU0255011-1.
- Figure 4.
Effect of voltage on [Cl–]i recovery. A, [Cl–]i recovery in an individual cell at two different levels of holding voltage, as indicated, in control solution. Note that [Cl–]i recovers quicker and to a lower concentration at –74 mV compared with at –14 mV. B, Summary of data showing [Cl–]i recovery at –74 and at –14 mV (as in A) in eight neurons. C, [Cl–]i recovery in an individual cell at two different levels of holding voltage, as indicated, in the presence of 10 µM VU0255011-1. D, Summary of data showing [Cl–]i recovery at –74 and at –14 mV in the presence of 10 µM VU0255011-1 (as in C) in five neurons. Probe pulses to estimate [Cl–]i were given at slightly different times during each recovery interval (to keep the membrane potential close to ECl). To compare at similar times, in B and D, interpolation was used for data pairs closest to the time points illustrated, and [Cl–]i was normalized to the maximal concentration during loading.
- Figure 5.
Relative resting Cl– conductance. A, Distribution of measured total conductances (in the presence of VU0255011-1, TTX, Cd2+, and TEA; see main text), showing no significant difference before and after Cl– loading, as required for the method used to estimate resting Cl– conductance. Data paired for individual cells. B, Relative resting Cl– conductance, in control conditions and in the presence of 100 µM PTX. Note that the given values in B are relative to the total membrane conductance in the absence of blockers.
- Figure 6.
Lack of ClC-2 currents. A, Voltage protocol (bottom) used for detecting ClC-2 currents and corresponding currents (top) recorded in an MPN neuron. Colors of individual current traces matches the colors in the voltage protocol. B, I-V relation for the currents in A (average amplitude during the time marked by olive bar in A). Note the small amplitude and the linearity in the range below –65 mV.
- Figure 7.
Role of spontaneously open GABAA receptors and of neurosteroid-potentiated mIPSCs for [Cl–]i recovery. A, Relative [Cl–]i, illustrating recovery 5 and 10 min after Cl– loading. Current-clamp (0 pA) between test ramps during recovery phase: control only. Voltage-clamp (–74 mV) between test ramps during recovery phase: control, 2.0 µM TTX, 3.0 µM gabazine, a combination of TTX and gabazine, or 10 µM strychnine, as indicated. Note the lack of significant differences. B, Relative [Cl–]i (as in A, voltage-clamp only), with significantly reduced recovery in the presence of 100 µM PTX. C, Relative [Cl–]i (as in B), showing gabazine-sensitive enhancement of recovery in the presence of 2.0 µM TTX and the neurosteroid allopregnanolone (Allo) in a concentration (20 nM) known to enhance mIPSC frequency and prolong mIPSC decay. D, Relative [Cl–]i (as in B) showing the dramatic enhancement of [Cl–]i recovery by allopregnanolone in a concentration (1.0 µM) that may directly activate GABAA receptors and the block of this effect by 100 µM PTX. E, Comparison of the effects of 20 nM Allo, DS2 and a combination of Allo and DS2 on normalized [Cl–]i after 5 min of recovery from a Cl– load as in A–D. Note the highly significant effect of DS2 and the additive effects of Allo and DS2. To compare data recorded at slightly different times in A–E, interpolation was used for data pairs closest to the time points illustrated and [Cl–]i was normalized to the maximal concentration during loading.
- Figure 8.
Computed [Cl–]i recovery. Dependence on gCl, gKCC2, and Vm. A, Recovery mediated by KCC2 only (red, middle), Cl– leak only (blue, top), or a combination of KCC2 and Cl– leak (black, bottom) at Vm = –74 mV. B, Recovery mediated by KCC2 and Cl– leak in combination at different Vm, as indicated. Note that the rate of recovery as well as the asymptotic steady-state [Cl–]i depends on Vm. C, Recovery mediated by KCC2 and Cl– leak in combination at different relative gCl, as indicated and at Vm = –74 mV (Ca) or Vm = –50 mV (Cb). Note that the speed of recovery increases with gCl and that the asymptotic steady-state [Cl–]i also increases with gCl. The latter effect is weak at –74 mV (Ca) but prominent at –50 mV (Cb). The transport capacity of KCC2 in (A–C) was adjusted by setting the transporter proportionality factor (apparent conductance; Johansson et al., 2016) gKCC2 to 6.7 10−21 mol2 V−1 C−1 s−1, to match modeled KCC2-mediated [Cl–]i recovery time course to experimental results.
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