Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha(1A)-subunit

Abstract

The Ca(2+) channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca(2+) channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. alpha(1A)-Subunits are thought to support both P- and Q-type Ca(2+) channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca(2+) delivery system at excitatory nerve terminals. We generated alpha(1A)-deficient mice (alpha(1A)(-/-)) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca(2+) channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca(2+) entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A).

Figure 1

Characterization of the mouse Ca²⁺ channel α_1A null mutation. (a) The start and the stop codons are indicated in the cDNA map. The solid box and the hatched box represent the corresponding exons in the genomic DNA locus. Double arrowhead lines under the wt and the targeted locus represent the expected fragments when digested with EcoRI and hybridized with the probe (open box underneath the wt locus). B, BalI; E, EcoRI; H, HindIII; X, XbaI; XI, XhoI. (b) Southern blot analysis of offspring derived from intercrosses of calcium channel α_1A heterozygous parents. The 9.4-kb fragment is the wt allele and the 9.0-kb fragment is the targeted allele. +/+, wt; +/−, heterozygote; −/−, mutant. (c) Northern blot analysis of brain tissues. One-microgram aliquots of poly(A) RNA from wt and −/− animals were probed with a 410-bp fragment from the 5′ region of rat brain α_1A cDNA. The lower bands are β-actin mRNA, used as a control. (d) Western blot analysis of membrane fractions from cerebral (Cr) and cerebellar (Cl) cortices of wt and α_1A^−/− mice. The band observed near 100 kDa in α_1A^−/− is unlikely to correspond to the glycosylated, 95-kDa short form of α_1A (19) because it was eliminated after purification by affinity to wheat germ-agglutinin-Sepharose. Furthermore, no reverse transcription–PCR products were obtained from α_1A^−/− mRNA with various primer sets targeting the first half of the channel, in contrast to the finding of PCR products of correct length when wt mRNA was tested (see Methods). (e) An α_1A mutant mouse attempting to right itself during a falling episode.

Figure 2

Histological and immunohistochemical analyses of brain in wt and α_1A^−/− mouse at P21. (a and b) Toluidine blue-stained sagittal sections of cerebellar vermis. (c and d) Higher magnification of areas in a and b. The mutant showed the persistence of an external granule cell layer, indicating a delay or deficit in granule cell migration (d). (e– h) Cerebellar cortex immunostained with anticalbindin antibody. (e and f) Dendritic trees of Purkinje cells. (g and h) Axons of Purkinje cells in granule cell layer. Arrow indicates focal axonal swellings that frequently are found in α_1A^−/− (h Inset). EGL, external granule cell layer. [Bars = 400 μm (a and b), 25 μm (c– h), and 5 μm (h Inset).]

Figure 3

P/Q-type calcium channels are absent and R-type calcium channels are diminished in α_1A^−/− cerebellar granule cells. (a) Peak I_Ba, activated by 30-ms depolarizations from –90 mV to –10 mV every 10 s, is plotted against time for an α_1A^+/+ neuron. The total current was reduced by the application of nimodipine (10 μM), ω-CTx-GVIA (1 μM), and ω-CTx-MVIIC (5 μM), which blocked the L-, N-, and P/Q-type components of the current, respectively. Representative traces are shown to the right of the graph. Ba²⁺ (10 mM) as charge carrier. (b) Peak I_Ba, activated as in a, is plotted against time for an α_1A^−/− neuron. (c) Histogram of total and individual channel type current density in α_1A^+/+ (solid), α_1A^+/− (hatched), and α_1A^−/− (open) granule cells. The total current density is reduced in α_1A^−/− neurons; this is accounted for by the absence of the P/Q-type channels and the reduction of R-type current. **, P < 0.002, *, P < 0.05.

Figure 4

P-type currents are eliminated and L- and N-type currents are augmented in Purkinje neurons from α_1A^−/−. (a and b) Pharmacological dissection of Ca²⁺ channel currents from α_1A^+/+ (a) and α_1A^−/− Purkinje neurons (b). Ba²⁺ (2 mM) as charge carrier. (Left) Response to successive application of 500 nM ω-Aga-IVB (purified native toxin), 1 μM nimodipine, and 1 μM ω-CTx-GVIA. (Right) Representative traces. (c) Current density distribution. Null mutants exhibited significantly smaller total current density than the controls. P-type currents were absent in α_1A^−/−. L- and N-type current densities were increased significantly in α_1A^−/−, whereas the other CdCl₂-sensitive components did not change. **, P < 0.001; *, P < 0.05. (Inset) Total current (Total) and Aga IVB-sensitive current (“P”) after preblockade of L- and N-type currents with nimodipine and ω-CTx-GVIA. Note that Aga IVB showed no effect on the remaining currents in α_1A^−/−. Thus, the modest Aga IVB effect on the null mutant neuron currents (b) can be attributed to a slight inhibition of N- or L-type currents (43).

Figure 5

Effects on synaptic transmission of altering Ca²⁺ influx in wt (●) and mutant (○) animals. (a) Blockade of N-type Ca²⁺ channels with ω-CTx-GVIA (1 μM) reduced the strength of synaptic transmission by half in wt but eliminated the EPSP in mutants. (b) Enhancement of Ca²⁺ influx by broadening action potentials with 4-AP (100 μM) increased the strength of synaptic transmission by 2-fold in wt and 3-fold in mutants. (c) After application of 4-AP, blockade of N-type Ca²⁺ channels considerably reduced synaptic strength in α_1A^−/− slices, with a much smaller effect in wt slices. (d) After application of 4-AP and ω-CTx-GVIA, blockade of P/Q-type Ca²⁺ channels with ω-Aga-IVB (1 μM) decreased the strength of synaptic transmission in wt by more than three-quarters but had no effect in mutants. (e and f) Representative responses to sequential application of Ca²⁺ channel blockers in the presence of 4-AP in wt (e) and α_1A^−/− (f) animals.