Leaky sodium channels from voltage sensor mutations in periodic paralysis, but not paramyotonia

Abstract

Background: Hypokalemic periodic paralysis (HypoPP) is associated with mutations in either the Ca(V)1.1 calcium channel or the Na(V)1.4 sodium channel. Some Na(V)1.4 HypoPP mutations have been shown to cause an anomalous inward current that may contribute to the attacks of paralysis. Herein, we test whether disease-associated Na(V)1.4 mutations in previously untested homologous regions of the channel also give rise to the anomalous current.

Methods: The functional properties of mutant Na(V)1.4 channels were studied with voltage-clamp techniques in an oocyte expression system.

Results: The HypoPP mutation Na(V)1.4-R1132Q conducts an anomalous gating pore current, but the homologous R1448C mutation in paramyotonia congenita does not.

Conclusions: Gating pore currents arising from missense mutations at arginine residues in the voltage sensor domains of Na(V)1.4 are a common feature of HypoPP mutant channels and contribute to the attacks of paralysis.

Figure 1. Mutations of the sodium channel (Na_V1.4) or calcium channel (Ca_V1.1) associated with periodic paralysis

Diagrams show the membrane folding topology and location of missense mutations associated with periodic paralysis. Three allelic disorders with periodic paralysis occur in Na_V1.4 (paramyotonia congenita [PMC], hyperkalemic periodic paralysis[HyperPP], hypokalemic periodic paralysis [HypoPP]). HypoPP mutations (red triangles) occur at arginine residues (R) in the voltage-sensor domains of Na_V1.4 or Ca_V1.1. The Na_V1.4 mutations investigated in this study are underlined.

Figure 2. Currents recorded from voltage-clamped oocytes expressing Na_V1.4 constructs

(A) The currents elicited by a series of step depolarizations of 400 msec duration over a range of −140 mV to 40 mV, in 10-mV increments from a holding potential of −100 mV. No leak subtraction has been performed; rapid transients are clipped. Only the HypoPP mutant R1132Q (black, middle trace) has large inward currents elicited at negative test potentials. Notice the R1132Q current offset at the holding potential of −100 mV. Wild-type (blue) and PMC R1448C mutants (red) have only small background leakage currents. (B) Current responses for a series of brief 15-msec depolarizations from −140 mV for the same oocytes. The linear background currents (leakage and capacitance) have been subtracted digitally to yield the gating charge displacement current. The area under the rapid current transient is an index of channel expression, and these data show that all 3 Na_V1.4 constructs had comparable levels of expression at the surface membrane.

Figure 3. Gating pore current for the hypokalemic periodic paralysis (HypoPP) R1132Q mutant

(A) Steady-state current recorded at the end of a 400-msec pulse for test potentials between −140 mV and +30 mV. The HypoPP R1132Q mutant passes an inward gating pore current at membrane potentials less than −60 mV (inward rectification), whereas WT and the paramyotonia congenita (PMC) R1448C mutant do not (n = 4 oocytes for WT and 5 for each mutant). Linear background currents have been subtracted by fitting the I-V response between −20 mV and +20 mV. (B) Selectivity of theR1132Q gating pore by measuring currents for a variety of test cations. The permeability sequence is K⁺> Na⁺> NMDG⁺ (N-methyl-d-glucamine). Under physiologic conditions, the primary charge carrier would be inward movement of Na⁺ ions.

Figure 4. Calcium does not block the R1132Q gating pore

Hypokalemic periodic paralysis (HypoPP) R1132Q gating pore currents were recorded in high external K⁺ (100 mM) as the Ca²⁺ was varied from 0.2 to 10 mM. Each oocyte served as its own control for expression level, as exposure to all 4 Ca²⁺ levels was performed and current amplitudes were normalized to the response at a test potential of −110 mV in 1.5 mM Ca²⁺ (n = 3 oocytes). The rightward shift of the I-V curve for higher [Ca²⁺] is caused by an apparent shift in the voltage dependence of channel gating due to screening of surface charges on the plasma membrane.

Figure 5. Acetazolamide (ACTZ) does not block gating pore currents in Na_V1.4 hypokalemic periodic paralysis (HypoPP) mutants

Current-voltage plots show exposure to 100 μM acetazolamide did not block the gating pore current for the HypoPP mutants Na_V1.4-R1132Q (A) or Na_V1.4-R672G (B). The current-voltage relation for the R672G mutant did not show saturation as we reported before, because the oocyte was maintained at a holding potential of −30 mV between test pulses.