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. 2006 Jul 27;576(Pt 3):739–754. doi: 10.1113/jphysiol.2006.115105

Figure 5. Charge reversal facilitates slow inactivation.

Figure 5

The voltage-clamp protocols are provided as insets in Figure 2. A, steady state fast-inactivation channel availability was not changed by substitution of external ring residues with arginine. The data were fitted as described in Fig. 2. The midpoints and slope factors were −55.5 ± 1.5 and 4.5 ± 0.3 E403R (n = 6), −55 ± 1.3 and 4.5 ± 0.3 E758R (n = 5), −54.2 ± 1.2 and 5.2 ± 0.2 D1241R (n = 15) and −55.5 ± 1.0 and 5.3 ± 0.1 D1532R (n = 7); compared with −53.0 ± 1.2 and 6.0 ± 0.3 WT, respectively. B, recovery from fast inactivation was unaltered by charge reversal. The time constants were 2.6 ± 0.2 (E403R), 2.5 ± 0.5 (E758R), 2.5 ± 0.1 (D1241R) and 2.5 ± 0.3 (D1532R), n = 4–5; compared with 3.0 ± 0.3 (WT). C, development of slow inactivation was significantly enhanced. The data were fitted in each case by a single exponential, n = 4–6. The time constants and fractional weights (in parentheses) were 3852 ± 1564 ms** (0.7 ± 0.02) for E403R, 4564 ± 1294 ms** (0.7 ± 0.0) for E758R, 7998.54 ± 1959.13 ms** (0.81 ± 0.02) for D1241R, 6898 ± 1009 ms** (0.8 ± 0.03) for D1532R; compared with 15612 ± 910. ms and 0.1 ± 0.02 (WT). D, charge reversal further impaired the kinetics of recovery from slow inactivation. The data were best fitted with two-order time constants. The time constants and fractional weight were: τ1 = 59.2 ± 24.8 ms (0.3 ± 0.03), τ2 = 3276 ± 1173 ms** (0.5 ± 0.02) for E403R (n = 5); τ1 = 33.8 ± 13.5 ms (0.4 ± 0.04), τ2 = 2627 ± 152 ms** (0.5 ± 0.04) for E758R (n = 4); τ1 = 17.4 ± 6.7 ms (0.4 ± 0.05), τ2 = 2640 ± 521 ms** (0.5 ± 0.04) for D1241R (n = 3); τ1 = 169 ± 94.4 ms (0.3 ± 0.03), τ2 = 5077 ± 942 ms** (0.7 ± 0.04) for D1532R (n = 6); compared with τ1 = 48.3 ± 37.1 ms (0.34 ± 0.10), τ2 = 407 ± 87.5 ms (0.7 ± 0.1) for WT (n = 5). E, E758R and E1532R mutant channels exhibited extra delay in recovery from slow inactivation compared with E758C and E1532C, respectively (P < 0.05). F, comparison of the effects of E403R and E1241R mutations on the recovery from slow inactivation with those of E403C and E1241C substitution, respectively (n.s.). *P < 0.05, **P < 0.01.