Figure 1.
GABAA receptor, ELIC, and GLIC current responses measured using two-electrode voltage clamp.
A) Representative currents from the GABAA receptor, ELIC, and GLIC expressed in oocytes evoked by saturating concentrations of agonist (black bar). B) Concentration response curves for the GABAA receptor, ELIC, and GLIC. CA = cysteamine.
Figure 2.
Outside-out patch-clamp recordings from HEK-293T cells expressing GABAARs.
A) Current evoked by a 1 second pulse of 10 mM GABA. The current decrease in the presence of agonist (desensitization) and upon agonist washout (deactivation) were fit with bi-exponential functions (red curves) and the weighted time constants for the example trace are shown. B) Current evoked by a 5 millisecond application of 10 mM GABA (black arrow). To the right, expanded traces highlight the current increase during GABA application (activation) and the current deactivation following washout of GABA. The time course of activation was determined by fitting the rising phase with a bi-exponential function (red curve) and the timecoure of deactivation was determined by fitting the current decay after agonist removal with a bi-exponential function (red curve). The weighted time constants for the example traces are shown. The traces shown are the ensemble averages of multiple GABA-evoked currents from a single patch.
Figure 3.
Cysteamine activation of ELIC.
Outside-out patch-clamp recordings from HEK-293T cells expressing ELIC. ELIC currents evoked by alternating pulses of 30 mM and 10 mM or 50 mM cysteamine (black bar) are overlaid. Current amplitudes and rise-times evoked by 30 mM and 50 mM cysteamine were similar suggesting 30 mM was saturating.
Figure 4.
ELIC current activation was slow and deactivation fast compared to GABAARs.
A) Macroscopic ELIC current evoked by a 2 s application of 30 mM cysteamine (black bar) (left). Expanded views depict current activation (center) and deactivation (right) fit with single exponential functions (red). B) The τ-activations for ELIC currents (30 mM cysteamine) and GABAAR currents (10 mM GABA) are plotted. Data are mean ± SEM (ELIC: n = 9, GABAAR: n = 19). C) The τ-deactivations for ELIC currents evoked by short (2s) and long (25s) pulses of 30 mM and 10 mM cysteamine are plotted. The τw-deactivations for GABAAR currents evoked by 5 ms or 1 s applications of 10 mM GABA are also plotted. GABAAR current deactivation after a 1 s pulse is significantly slower than after a 5 ms pulse (* p<0.001). Data are mean ± SEM (ELIC 30 mM 2 s: n = 9, ELIC 30 mM 25 s: n = 3, ELIC 10 mM 2 s: n = 6, ELIC 10 mM 25 s: n = 4, GABAAR 5 ms: n = 9, GABAAR 1 s: n = 10).
Figure 5.
Macroscopic ELIC current desensitization.
A) The desensitization phase of ELIC current evoked by a 25 s pulse of 30 mM cysteamine was fit with a single-exponential function (red curve). B) The τ-desensitization for ELIC currents as measured in (A) and the τw-desensitization for GABAAR currents measured during a 1s application of 10 mM GABA are plotted. Data are mean ± SEM (ELIC: n = 4, GABAAR: n = 10).
Figure 6.
ELIC currents evoked by GABA exhibited unique kinetics.
A) ELIC currents evoked by GABA (25 s, 100 mM) exhibited slow current activation, which was fit with a bi-exponential function (red curve). B) Following activation by a 1 s pulse of 100 mM GABA, ELIC current decayed very slowly and was fit with a bi-exponential function (red curve). Each GABA-evoked trace (black) is overlaid with a normalized response to 30 mM cysteamine obtained from a separate patch (gray) to highlight the differences in current activation and deactivation kinetics.
Figure 7.
A) GLIC currents evoked by alternating 2 s jumps from pH 7.6 to the indicated values. Maximal current amplitudes were evoked by pH 4.0. B) A current recorded from an outside-out patch pulled from a mock-transfected HEK-293T cell evoked by pH 4.0 (green) is normalized and overlaid with GLIC currents (black) evoked by pH 4.0 from outside-out patches pulled from two different HEK-293T cells expressing GLIC to highlight differences in macroscopic current desensitization.
Figure 8.
GLIC current activation was slow and deactivation fast compared to GABAARs.
A) GLIC currents activated by a 2 s application of pH 4.0 buffer exhibited two different desensitization profiles. In 25 patches, little current desensitization was observed, whereas in 18 patches, a fast component of desensitization was observed. GLIC current activation and deactivation was analyzed separately for each group. Expanded views of current activation and deactivation are shown to the right and fit with bi-exponential functions (red curves). B) The τw-activations for GLIC currents (pH 4.0) from both groups are plotted and compared to the τw-activations of currents obtained from patches from mock-transfected cells (pH 4.0) and from GABAARs (10 mM GABA). Data are mean ± SEM (GLIC no early: n = 25, GLIC w/early: n = 18, Mock: n = 7). C) The τw-deactivations are plotted for both groups of GLIC currents following a 2 s (as shown above) as well as a 25 s pulse of pH 4.0 buffer. The τw-deactivations for GABAAR currents evoked by 5 ms or 1 s applications of 10 mM GABA are also plotted. Data are mean ± SEM (GLIC no early 2 s: n = 11, GLIC no early 25 s: n = 8, GLIC w/early 2 s: n = 6, GLIC w/early 25 s: n = 6).
Figure 9.
GLIC current desensitized slowly.
A) GLIC current desensitization during a 25 s pulse of pH 4.0 buffer was fit with a single exponential when there was no early desensitization (left trace, red curve) or a bi-exponential function when early desensitization was present (right trace, red curve). B) Time constants for the fast and slow components of desensitization from both types of GLIC currents (pH 4.0, 25 s) and GABAAR currents (10 mM GABA, 1 s) are plotted. n.a. = not applicable, no measurable fast component. C) The τw-desensitizations for both types of GLIC currents (pH 4.0, 25 s) and the GABAAR (10 mM GABA, 1 s) are plotted. Data are mean ± SEM (GLIC no early: n = 7, GLIC w/early: n = 9, GABAAR: n = 10). D) GLIC current desensitization during a 150 s pulse of pH 4.0 buffer was fit with a single exponential when there was no early desensitization (left trace, red curve) or a bi-exponential function when early desensitization was present (right trace, red curve). E) GLIC current decayed to baseline during an 8 minute pulse of pH 4.0 buffer.