Table 1. V1/2 of channel open probability (Po), fast gating (Pofast) and common gating (Pocommon) for truncations, alanine substitutions and co-expression of N1–720 with short peptides.
Peak tail currents were used to derive Po values and fast and common gating parameters as described in the Experimental section. Measurements are given in mV as mean±S.E.M. Truncations: the overall Po curve of the truncation N1–885 (removal of the final 103 residues) was approx. 45 mV more positive than WT, and Pofast and Pocommon were some 30–40 mV more positive than WT as revealed by V1/2 values. For truncation N1–888 (the removal of the final 100 amino acids), the V1/2 values for the Po, Pofast and Pocommon curves were similar to WT although the minimum value of Pocommon was larger (Figure 1). Alanine substitutions: the results of alanine substitution for Glu865 in the co-expression N1–720+C721–988(E865A) or of both Glu864 and Glu865 in N1–720+C721–988(E865A, E865A) were different from all the other mutant currents, as the overall Po curves were shifted towards more negative voltages. Fast gating was similar to WT, and the entire shift in Po could be attributed to common gating. The Po, Pofast and Pocommon curves for alanine substitutions of Glu864 alone in N1–720+C721–988(E864A) and of Leu866, Gln867 and Lys868 in N1–720+C721–988(L866A, Q867A, K868A) were similar to WT. Co-expression of G721X with short peptides: co-expression of carboxyl tail truncation N1–720 with C863–888 (26 amino acids) or C863–900 (38 amino acids), which are missing both the 142 amino acids between CBS1 and CBS2 and the final 100 or 88 amino acids respectively, gave smaller chloride currents but the Po curves were similar to WT.
| RNA (no. of oocytes) | V1/2 of Po (mV) | V1/2 of Pofast (mV) | V1/2 of Pocommon (mV) |
|---|---|---|---|
| WT hClC-1 (13) | −56.4±2.1 | −102.8±1.6 | −47.7±4.7 |
| N1–885 (10) | −11.9±1.0 | −67.3±3.8 | −10.0±5.4 |
| N1–888 (10) | −47.4±1.5 | −79.3±2.0 | −67.4±7.9 |
| N1–720+C863–988 (10) | −57.1±1.7 | −91.8±1.9 | −50.5±4.4 |
| N1–720+C863–900 (12) | −46.8±1.5 | −92.3±1.8 | −34.1±4.2 |
| N1–720+C863–888 (16) | −62.3±1.6 | −98.9±0.8 | −35.5±4.4 |
| N1–720+C721–988 (10) | −60.1±1.1 | −107.8±1.7 | −59.3±2.9 |
| N1–720+C721–988(E864A, E865A) (7) | −83.5±2.3 | −102.8±3.1 | −81.2±6.1 |
| N1–720+C721–988(L866A, Q867A, K868A) (13) | −52.7±1.7 | −111.1±2.5 | −34.8±7.7 |
| N1–720+C721–988(E864A) (10) | −69.6±2.2 | −111.6±2.1 | −44.9±8.6 |
| N1–720+C721–988(E864A) (11) | −75.7±1.9 | −100.7±1.6 | −75.1±7.6 |