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Case Reports
. 2000 Aug;106(3):431-8.
doi: 10.1172/JCI9654.

A double mutation in families with periodic paralysis defines new aspects of sodium channel slow inactivation

S Bendahhou  1 , T R Cummins, A F Hahn, S Langlois, S G Waxman, L J Ptácek
Affiliations
Case Reports

A double mutation in families with periodic paralysis defines new aspects of sodium channel slow inactivation

S Bendahhou et al. J Clin Invest. 2000 Aug.

Abstract

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant skeletal muscle disorder caused by single mutations in the SCN4A gene, encoding the human skeletal muscle voltage-gated Na(+) channel. We have now identified one allele with two novel mutations occurring simultaneously in the SCN4A gene. These mutations are found in two distinct families that had symptoms of periodic paralysis and malignant hyperthermia susceptibility. The two nucleotide transitions predict phenylalanine 1490-->leucine and methionine 1493-->isoleucine changes located in the transmembrane segment S5 in the fourth repeat of the alpha-subunit Na(+) channel. Surprisingly, this mutation did not affect fast inactivation parameters. The only defect produced by the double mutant (F1490L-M1493I, expressed in human embryonic kidney 293 cells) is an enhancement of slow inactivation, a unique behavior not seen in the 24 other disease-causing mutations. The behavior observed in these mutant channels demonstrates that manifestation of HyperKPP does not necessarily require disruption of slow inactivation. Our findings may also shed light on the molecular determinants and mechanism of Na(+) channel slow inactivation and help clarify the relationship between Na(+) channel defects and the long-term paralytic attacks experienced by patients with HyperKPP.

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Figures

Figure 1
Figure 1
Schematic of the voltage-gated skeletal muscle Na+ channel α-subunit showing the location of naturally occurring mutations described to date (mutations 1–19) and the double mutation (open circles) F1490L-M1493I described in this study. Mutations 1, 4, 6, 7, 8, and 18 are associated with PAM; mutations 2, 9, 12, 13, 14, 15, and 16 are associated with PC; and mutations 3, 5, 10, 11, 17, and 19 are associated with HyperKPP. The lower panel shows how the phenylalanine and methionine mutated are well conserved between different cloned Na+ channels: human skeletal muscle (hSkM1), human cardiac muscle (hSkM2), rat brain type II (RII), eel electroplax, squid giant axon, TTX-resistant Na+ channels from dorsal root ganglia (DRG), jellyfish, and Drosophila para.
Figure 2
Figure 2
Current traces from representative HEK 293 cells expressing either WT channels, F1490L, M1493I, or F1490L-M1493I mutant channels. Na+ currents were monitored in the whole-cell configuration and were elicited by repetitive 25-millisecond voltage steps ranging from –80 to 0 mV. The holding potential was –120 mV. pA, picoamperes.
Figure 3
Figure 3
Activation and inactivation curves. Peak Na+ conductance (GNa) was measured during a 25-millisecond depolarization to various test potentials from a holding potential of –120 mV to characterize steady-state activation for WT (filled circles; n = 47), F1490L (filled diamonds; n = 28), M1493I (filled squares; n = 21), and double mutant F1490L-M1493I (open circles; n = 24). GNais calculated from the relation GNa = INa/(V – Vrev), where INa is the peak inward Na+ current during the test depolarization (V) and Vrev is the Na+ reversal potential. Data are normalized to maximum peak conductance (Gmax) and fit to a two-state Boltzmann distribution: GNa/Gmax = (1 + exp[(V – V1/2)/k])–1, where V1/2 is the test potential for half-maximal Na+ activation and k determines the steepness of the voltage dependence. Inactivation curves plotted for Na+ currents for WT (filled circles; n = 43), F1490L (filled diamonds; n = 29), M1493I (filled squares; n = 19), and double mutant F1490L-M1493I (open circles; n = 34). Cells were held at –120 mV and subjected to a 200-millisecond conditioning pulse ranging from –120 to 0 mV followed by a 25-millisecond test pulse to 0 mV. Values are mean ± SEM.
Figure 4
Figure 4
Recovery from fast inactivation and deactivation. (a) Recovery from fast inactivation. Cells were prepulsed to 0 mV for 200 milliseconds to inactivate all of the current, then recovery potentials from –120 to –60 mV (–120 mV is shown) for increasing recovery duration were applied before the test pulse to 0 mV (20 milliseconds) to assay the fraction of current recovered. Traces obtained at the test pulse were fitted to a single-exponential function. (b) Tail currents were elicited by a 0.5-millisecond test pulse to +40 mV followed by a repolarization pulse ranging from –120 to –60 mV. Resulting currents were fitted by a single-exponential decay and expressed as a function of the voltage for WT (filled circles; n = 20), F1490L (filled diamonds; n = 20), M1493I (filled squares; n = 20), and F1490L-M1493I (open circles; n = 20). Values represent mean ± SEM.
Figure 5
Figure 5
Enhancement of slow inactivation in the F1490L-M1493I channels. (a) Steady-state slow inactivation in hSkM1 (filled circles; n = 23), F1490L (filled diamonds; n = 9), M1493I (filled squares; n = 9), and F1490L-M1493I (open circles; n = 18) channels. The first step consists of holding the cells at potentials ranging from –130 to 10 mV in 10-mV steps for 50 seconds. A 30-millisecond recovery pulse to –100 mV and a 20-millisecond test pulse to –10 mV were given before the holding potential was incremented again. The holding potential was incremented by 10 mV immediately after each recovery pulse/test pulse sequence. We term this protocol sequential because the channels are not allowed to recover from slow inactivation for each test pulse; the expectation is that the sequential changes in holding potential will mimic changes in holding potential of longer durations. The short hyperpolarizing recovery pulses can be used to remove fast inactivation just before a test pulse to measure slow inactivation (31). The peak current elicited by the test pulse to –10 mV was plotted as a fraction of the maximum current. (b) A greater fraction of F1490L-M1493I current than WT current is slow inactivated at –60 mV. Cells were held for 50 seconds at –60 mV, allowed to recover for 30 millisecond at –100 mV, and then depolarized to –10 mV to determine the fraction of channels that are slow inactivated. For comparison, the total current available from a holding potential of –100 mV is also shown for WT and F1490L-M1493I channels. (c) Development of slow inactivation is shown to be faster for the F1490L-M1493I (open circles; n = 5) than for the WT channels (filled circles; n = 5). Cells were held at –100 mV for an increasing conditioning time. (d) Normalized Na+ current in representative WT (filled circles; n = 9), F1490L (filled diamonds; n = 8), M1493I (filled squares; n = 7), and F1490L-M1493I (open circles; n = 8) cells recovering from slow inactivation (conditioning pulse is –50 mV). The time axis is logarithmic. The recovery protocol required about 45 minutes to complete and had two phases: short recovery times were obtained with individual recovery pulses, and long recovery times were obtained in a continuous recording.
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