Functional expression of the Ile693Thr Na+ channel mutation associated with paramyotonia congenita in a human cell line

Abstract

1. The Ile693Thr mutation of the skeletal muscle Na+ channel alpha-subunit is associated with an unusual phenotype of paramyotonia congenita characterized by cold-induced muscle weakness but no stiffness. This mutation occurs in the S4-S5 linker of domain II, a region that has not been previously implicated in paramyotonia congenita. 2. The Ile693Thr mutation was introduced into the human skeletal muscle Na+ gene for functional expression in human embryonic kidney (HEK) cells. The currents expressed were recorded with the whole-cell voltage-clamp technique. 3. In comparison with wild-type currents, Ile693Thr mutant currents showed a clear shift of about -9 mV in the voltage dependence of activation. 4. In contrast to other mutations of the Na+ channel known to cause paramyotonia congenita, the Ile693Thr mutation did not induce any significant change in the kinetics, nor in the voltage dependence, of fast inactivation. 5. In conclusion, this study provides further evidence of the involvement of the S4-S5 linker in the voltage dependence of Na+ channel activation. The negative shift in the voltage dependence found in this mutation must be associated to other defects, plausibly an impairment of the slow inactivation, to account for the long periods of muscle weakness experienced by the patients.

Figure 1. Voltage dependence of activation and fast inactivation kinetics for wild-type and Ile693Thr mutant Na⁺ channel recorded in 3 mm (A-C) or 30 mm (D-F) external K⁺

A and D, representative traces of wild-type and Ile693Thr mutant channels elicited at two different command potentials as indicated below the traces. Note the lower threshold for mutant channel activation than for wild-type. B and E, normalized current-voltage relationships obtained from HEK 293 cells expressing either wild-type (○, n = 7 and n = 5 in B and E, respectively) or Ile693Thr mutant channels (•, n = 8 and n = 5 in B and E, respectively); data are expressed as means ±s.e.m. C and F, voltage dependence of the fast inactivation time constant for wild-type (^) and mutant channels (•); data shown in C and F were respectively computed from the same recordings as those used in B and E.

Figure 2. Voltage dependence of steady-state inactivation and recovery from fast inactivation

^, wild-type channels; •, mutant channels. A, steady-state inactivation (fraction of maximum current) and activation curves (fraction of maximum conductance) are plotted together to show the window current, enlarged in B. C, time course of Na⁺ current recovery from fast inactivation for wild-type and mutant channels.

Figure 3. Temperature dependence of the steady-state current

Current-voltage relationship of the steady-state current measured at 20 ms of varying voltage steps for wild-type (^) and Ile693Thr mutant (•) channels. A, data obtained at 20–22 °C for wild-type (n = 6) and mutant channels (n = 6). B, data obtained at 13–15 °C for wild-type (n = 5) and mutant channels (n = 6). C, steady-state currents measured at 200 ms following voltage steps to −10 mV. Recordings were performed at room temperature or after cooling to 13–15 °C as indicated. At both command potentials, open columns represent data obtained for wild-type (n = 6 and n = 6, at 20–22 °C and 13–15 °C, respectively), and closed columns show data obtained for Ile693Thr mutant (n = 4 and n = 6 at 20–22 °C and 13–15 °C, respectively).

Figure 4. Current-voltage relationships (A) and corresponding activation curves (B) obtained for wild-type and mutant channels at a temperature of 13–15 °C

Note the negative shift in the voltage dependence of mutant (•, n = 6) compared with wild-type channels (^, n = 5).