Activation and inactivation of the voltage-gated sodium channel: role of segment S5 revealed by a novel hyperkalaemic periodic paralysis mutation

Abstract

Hyperkalaemic periodic paralysis, paramyotonia congenita, and potassium-aggravated myotonia are three autosomal dominant skeletal muscle disorders linked to the SCN4A gene encoding the alpha-subunit of the human voltage-sensitive sodium channel. To date, approximately 20 point mutations causing these disorders have been described. We have identified a new point mutation, in the SCN4A gene, in a family with a hyperkalaemic periodic paralysis phenotype. This mutation predicts an isoleucine-to-phenylalanine substitution at position 1495 located in the transmembrane segment S5 in the fourth homologous domain of the human alpha-subunit sodium channel. Introduction of the I1495F mutation into the wild-type channels disrupted the macroscopic current inactivation decay and shifted both steady-state activation and inactivation to the hyperpolarizing direction. The recovery from fast inactivation was slowed, and there was no effect on channel deactivation. Additionally, a significant enhancement of slow inactivation was observed in the I1495F mutation. In contrast, the T704M mutation, a hyperkalaemic periodic paralysis mutation located in the cytoplasmic interface of the S5 segment of the second domain, also shifted activation in the hyperpolarizing direction but had little effect on fast inactivation and dramatically impaired slow inactivation. These results, showing that the I1495F and T704M hyperkalaemic periodic paralysis mutations both have profound effects on channel activation and fast-slow inactivation, suggest that the S5 segment maybe in a location where fast and slow inactivation converge.

Fig. 1.

Alignment of the segment IV–S5 of the sodium channel. Segment S5 of domain IV deduced amino acid sequence of the voltage-gated sodium channel from human skeletal muscle (hSkM1), human cardiac muscle (hSkM2), rat brain type II (RII), eel electroplax, squid giant axon (Squid), TTX resistant from dorsal root ganglia (DRG), jellyfish, and Drosophilapara, along with isoleucine 1495-to-phenylalanine substitution. This figure shows that isoleucine 1495 is well conserved among voltage-gated sodium channels.

Fig. 2.

Effect of I1495F mutation on macroscopic currents. Cells were held at −120 mV, and inward sodium currents were elicited by 10 mV voltage steps from −80 to 20 mV for WT (A) and I1495F (B).C shows normalized current traces from WT and I1495F mutant recorded at −10 mV. D, An example of superimposed current recordings from WT and I1495F channels using the same pulse protocol as in A–C for a 100 msec duration.

Fig. 3.

Inactivation kinetics of I1495F. A, Time constant of fast inactivation for WT (filled circles, n = 39) and I1495F (open circles, n = 14) showing an additional slow component (open squares,n = 14) for the HyperKPP mutant channels.B, Steady-state fast inactivation. HEK 293 cells were subjected to a protocol described in Materials and Methods to generate the inactivation curves for WT (filled circles,n = 43) and I1495F (open circles,n = 31).

Fig. 4.

WT and I1495F activation parameters. The pulse protocol for constructing the current–voltage relationship is described in Materials and Methods. A, The current–voltage curves for WT (filled circles,n = 47) and I1495F (open circles,n = 31) were normalized for a better appreciation of the shift between the two relations. B, Currents were converted to conductance, and fit was made according to the Boltzmann function described in Materials and Methods. Symbols are the same as in A.

Fig. 5.

Deactivation, development, and recovery from inactivation for I1495F. A, Tail currents were elicited by a 0.5 msec test pulse to 40 mV, followed by repolarization pulses ranging from −120 to 0 mV. Resulting currents were fitted by a single-exponential decay and expressed as function of the voltage for WT (filled circles, n = 20) and for I1495F (open circles, n = 20). B, To assess the rate of recovery from fast inactivation, the following protocol was applied to HEK 293 cells: channels were fast inactivated during 200 msec using 0 mV voltage pulse, then they were allowed to recover at different voltages (−80 mV test pulse is shown as an example) for an increasing time, and finally a 0 mV test pulse was applied to test for the fraction of the channels recovered. Peak currents obtained using the test pulse were normalized to the peak current obtained during the inactivating pulse for WT (filled circles, n = 14) and I1495F (open circles, n = 26).C, Data for development of inactivation (squares) and recovery from fast inactivation (circles) were fitted to a single-exponential function and plotted as a function of the development–recovery pulse voltage. The averaged time constants from WT (filled symbols, n = 10) and I1495F (open symbols, n = 7) are shown.

Fig. 6.

I1495F enhances slow inactivation.A, Steady-state slow inactivation in hSkM1 WT (filled circles, n = 8) and I1495F (open circles, n = 8) after 50 sec conditioning pulse ranging from −130 to 10 mV. Band C show development of slow inactivation and recovery from slow inactivation, respectively, as a function of time for WT (filled circles, n = 7) and I1495F (open circles, n = 7) channels. Protocols are as described in Materials and Methods.

Fig. 7.

Effect of the T704M mutation on hSk6M1 currents. Family of traces from representative HEK 293 cells expressing either WT (A) or T704M (B) channels. The currents were elicited by 40 msec test pulses to various potentials from −60 to 30 mV. Cells were held at −100 mV. C, Peak current–voltage relationship for WT (filled squares, n = 8) and T704M (open squares, n = 7). D, The steady-state fast inactivation curves for WT (filled circles, n = 12) and T704M (open circles, n = 10) channels with 500 msec inactivating prepulses are shown. Cells were held at prepulse potentials over the range of −130 to 10 mV before a test pulse to −10 mV for 20 msec. Current is plotted as a fraction of peak current. Currents were converted to conductance, from the current–voltage curve shown in Figure 7C, for WT (filled squares, n = 8) and T704M (open squares, n = 7), and fit was made according to the Boltzmann function.

Fig. 8.

Deactivation, development, and recovery from fast inactivation for the T704M. The protocols used for the T704M are the same as those described for the I1495F mutation in Figure 5.A, Tail current traces obtained from WT and T704M channels at −70 mV. B, Time constants of tail current as shown in A were plotted as a function of the test potential for WT (filled circles,n = 5) and T704M (open circles,n = 6). C, Development of fast inactivation (squares) and recovery from fast inactivation (circles) for WT (filled symbols, n = 8) and T704M (open symbols, n = 7) were obtained as described in Figure 5.

Fig. 9.

Slow inactivation is impaired in T704M cells. Changes in peak sodium current for representative WT (A) and T704M (B) cells in response to changes in the holding potential. Cells were allowed to stabilize for 20 min at −80 mV before beginning the test protocol. The protocol involved holding the cell at a specific potential for 5 min, while once every 15 sec the membrane potential was stepped to −100 mV for 30 msec and then to 0 mV for 20 msec. The peak current elicited by the test pulse to 0 mV is plotted. C, The steady-state slow inactivation curves for WT (filled circles,n = 8) and T704M (open circles,n = 7) cells are shown. D, Normalized Na current in representative WT (filled circles, n = 5) and T704M (open circles, n = 6) cells recovering from slow inactivation. The time axis is logarithmic. The recovery protocol required ∼45 min to complete and had two phases: short recovery times were obtained with individual recovery pulses, long recovery times were obtained in a continuous recording. E, Development of slow inactivation is shown to be slower for the T704M (open circles, n = 5) than for the WT channels (filled circles, n = 5). Cells were held at −100 mV for an increasing conditioning time. Details of the protocol are described in Materials and Methods.