Mutation I810N in the alpha3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

Abstract

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na(+),K(+)-ATPase alpha3 isoform inactive. Total Na(+),K(+)-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na(+),K(+)-ATPase alpha3 by transgenesis, which also rescued Na(+),K(+)-ATPase activity. Our findings reveal the functional significance of the Na(+),K(+)-ATPase alpha3 isoform in the control of epileptiform activity and seizure behavior.

Fig. 1.

Positional cloning of Myshkin. (A) Fine mapping of the Myshkin mutation on Chr 7. White squares represent 129S1:B6 heterozygous alleles and black squares represent homozygous B6 alleles. The number of progeny inheriting each haplotype is listed on the bottom line. (B) DNA sequence traces of Atp1a3 exons 4 and 18 from Myshkin mice and the 129S1 and B6 parental strains. Asterisks indicate the location of a T→A transversion at nucleotide 328 and a T→A transversion at nucleotide 2,562. The sequences of Atp1a3 exons 4 and 18 in the mapping strains 129S1 and B6 are identical, indicating that 328T→A and 2562 T→A are de novo mutations. (C) EcoO109I restriction site (5′-AG*GGCCT-3′) generated by the 328T→A mutation in Atp1a3. A 576-bp PCR amplicon is cleaved by EcoO109I into 212-bp and 354-bp fragments. (D) Alignment of partial α3 protein sequences of several vertebrate species (Upper) and partial Mus musculus α1, α2, and α3 protein sequences (Lower), showing conservation of amino acids D65 (Blue text) and I810 (Red text).

Fig. 2.

Epileptic seizures in Myk/+ mice. (A) Spontaneous seizure in 16-week-old Myk/+ N2 B6 male. Frames 1–4 were captured at 4-s intervals after the loss of postural control. (B) Electrocorticogram trace from Myk/+ mouse before (Preinduction) and after cage shaking (Postinduction). (C) Number of electrographic seizures observed in +/+ (n = 6), Myk/+ (n = 9, **, P < 0.005), and Myk/+/BAC (n = 6) mice after cage shaking.

Fig. 3.

Reduced threshold for epileptiform activity in Myk/+ mice. (A) Bright-field image of transverse slice of hippocampus and entorhinal cortex (EC). Positions of the stimulating electrode (S) and of the recording electrodes (R) are illustrated. (B) Time to start of quasi rhythmic spiking after washout of extracellular Mg²⁺ (*, P < 0.05, **, P < 0.005, t test). (C) Response incidence for different types of epileptiform activity (illustrated in D and E) within 1 h of Mg²⁺ washout. “Sync” refers to the incidence of synchronized ictal bursts in EC and CA3 within 2 h of Mg²⁺ washout (**, P < 0.005, χ²-test). (D) Sample sweeps illustrating repeating synchronized ictal bursts in Myk/+ slice. Below is shown a single ictal burst on an expanded time scale. (E) Typical sample sweeps from +/+ slice.

Fig. 4.

Enhanced E-S coupling (popAP:fEPSP ratio) in Myk/+ mice, 25–55 min after TBS. (A) Before TBS, the relation between the postsynaptic fEPSP slope in CA1 stratum radiatum and the popAP, measured at the same transverse location in CA1 stratum pyramidal, did not differ between genotypes (“baseline”, open symbols). After TBS, Myk/+ mice exhibited a higher E-S coupling (popAP:fEPSP slope) than +/+ mice. (B) Summary of the E-S coupling data in A (*, P < 0.001, **, P < 10–9, #P < 0.01).

Fig. 5.

Mutational and BAC transgenesis effects on Na⁺,K⁺-ATPase activity and α3 expression in brain. (A) Specific Na⁺,K⁺-ATPase activity in brain homogenates of 10-week-old Myk/+ (n = 6) and tm1Ling/+ mice (n = 4) and of Myk/+ (n = 11) and Myk/Myk E18 fetuses (n = 5) (mean ± SEM). Results are expressed as a % of +/+ littermate levels (adults n = 6, fetuses n = 7). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus respective +/+ littermates; ###P < 0.001 versus tm1Ling/+ mice. (B) Specific Na⁺,K⁺-ATPase activity in brain homogenates of 10-week-old Myk/+ (n = 4) and Myk/+/BAC (n = 4) mice (mean ± SEM). Results are expressed as a % of +/+ littermate levels. **, P < 0.01 versus Myk/+ mice; ####P < 0.0001 versus +/+ mice. (C) Typical blots of 20 μg protein from brains probed with anti- Na⁺,K⁺-ATPase α3, anti-NR1, and anti-β-tubulin III antibodies (Left). Immunoreactivity of Myk/Myk (n = 4) and Myk/+ (n = 6) E18 fetus brains and Myk/+ (n = 8) adult brains expressed as a percentage of +/+ (E18 n = 4; adult n = 8) levels (Right). Each sample was blotted 3 times, and β-tubulin III was used as loading control. *, P < 0.05 versus +/+ E18 fetus brains (Right). (D) Distribution and signal intensity of α3 (Red) and agrin (Green) in cultured neocortical neurons from E18 fetuses (Left). (Scale bar, 24 μm.) Colocalization coefficients for Myk/Myk (n = 14) and +/+ (n = 14) E18 fetuses (Right).