Legends for supplementary figures
Figure 1. a. Peak calcium current was measured as a function of time prior and during application of 100 M GABA in the presence of the GABAA receptor antagonist bicuculline (100 M). Solid bar represents transmitter application. b. Effect of recombinant RGS12 on the magnitude of GABA-mediated inhibition of calcium current. Histogram shows the effect of injection of 2 nM RGS12 or control internal solution on the magnitude of voltage-dependent and –independent inhibition. Data represent mean values + S.E.M. of 5 independent experiments. c. Current-voltage relationship for control and antibody-injected cells. Currents were measured at the indicated voltages stepping from –80 mV. Prepulse was applied prior to the test pulse to measure voltage-independent inhibition.
Figure 2. a. Time course of the GABA-mediated voltage-independent inhibition in the presence of fusion proteins containing the PTB ands PDZ domains from RGS12. b.Traces showing calcium currents evoked by a test pulse from –80 mV to 0mV in cells equilibrated for 10 minutes with control or PTB-containing internal solutions. Traces marked as “a” were measured prior to transmitter application; traces “b” were recorded in the presence of 100 M GABA+ bicuculline; traces marked as “c” were obtained after 45 seconds under the constant presence of transmitter.
Figure 3. a. Immunoblotting of the membrane fraction from a DRG neuron cell lysate using antibodies against the 1B of the calcium channel. The band of the expected size is at about 240 kDa and is marked with an arrow. b. Immunoblotting with anti-1B calcium channel antibody raised against rat amino acid sequence 851-867 detects of a fusion protein containing the chick sequence for the same region. A GST-fusion protein containing the chick sequence for aa 721-963 loop II-III from embryonic chick sensory neurons was recognized by the anti-alpha 1B antibody raised against rat aa 851-867 (- lane). Preincubation of the antibody (1:1, 16 hours at 4 C) with a peptide encoding this sequence blocked the reactivity of the antibody with the fusion protein (+ lane). c. Immunoblot of immunoprecipitates using a second antibody raised against aa 1382 to 1400 of the rat
1 subunit from skeletal muscle. This region is conserved across all the 1 subunits of high-voltage activated calcium channels.
Table 1. Effect of antibodies raised against RGS proteins on the rate of desensitization of GABA-mediated steady-state inhibition
Antibody (200 ng/ml) / # of cells / t 1/2 (s)control / 10 / 42 ± 8
anti-RGS12 / 7 / 605 ± 14
anti-RGS4 / 4 / 43 ± 7
anti-GAIP / 5 / 41 ± 6
anti-RGS10 / 4 / 43 ± 8
Table 2. Effect on the rate of desensitization of fusion proteins containing various domains of RGS12. This table summarizes the data for all the proteins tested. Species abbreviations: h, human; m, mouse; r, rat. Amino acid numbering corresponds to SwissProt accession numbers O08774 (rat RGS12), O14924 (human RGS12).
Fusion protein / # of cells / t 1/2 (s)Control / 15 / 42.5 + 7
hRGS12PDZ domain (aa 1-110) / 10 / 45.2+ 4
rRGS12PDZ domain (aa 1-99) / 12 / 43.7 + 3
hRGS12PTBdomain (aa220-366) / 9 / 592 + 8
rRGS12PTB domain (aa193-394) / 8 / 586 + 5
hRGS12PTB and PDZ domain (aa 1-440) / 5 / 597 + 4
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