Article Figures & Data
Figures
- Fig. 1.
Generation of a mouse with a disrupted allele of the Cacna1e gene. A, Targeting of theCacna1e gene is shown. Top, The genomic structure of the wild-type allele of the Cacna1e gene is shown with exons indicated by black boxes. Restriction sites used for determination of the disrupted allele are indicated.Bottom, A PGK neolURA3 expression cassette replaced the fourth through eighth exons of this fragment. The locations of probes used for genomic blot hybridization are shownbelow the disrupted allele. B, Genomic blot hybridization of DNA derived from neomycin-resistant ES cell clones is shown. ES cell DNA was probed with a 3′-flanking probe (left) and 5′ internal probe (right). Hybridization with the 3′ probe shows a 6 kb wild-type band and 6.6 kb mutant band, whereas the 5′ internal probe revealed an 8.7 kb wild-type band and 12 kb mutant band. C, Tail tip DNA from three intercross littermates was probed with the 3′-flanking probe and showed the expected size for wild-type, heterozygous, and homozygous mutant.ko, Knock-out.
- Fig. 2.
Western blot analysis of α1E protein in wild-type and α1E-deficient mice. Brain lysates from 129/SV and C57BL/6J wild-type mice show the expression of the 230 kDa α1E protein, whereas the protein is clearly absent from brain tissue of α1E KO mice. Specificity of the antibody was confirmed by detection of a band in α1E-transfected HEK cell membranes and the lack of staining in untransfected HEK cell membranes.
- Fig. 3.
Effects of drugs and toxins on the cerebellar granule cell IBa. A, Plot of the peak IBa versus time in an α1E KO mouse shows a resistant component of the high voltage-activated IBa remaining after consecutive bath application of specific drugs and toxins. SNX-482 had no effect on the toxin-resistant current. Inset,Representative traces at numbered points are shown. B, Plot of the peakIBa versus time from a wild-type C57BL/6J mouse is shown. Inset, Representativetraces are shown. SNX-482 inhibited a component of the R current remaining after nimodipine, ω-agatoxin IVA (ω-Aga IVA), and ω-conotoxin GVIA (ω-CTx GVIA) application. C, Plot of the peakIBa versus time from a wild-type 129/SV mouse is shown. SNX-482 also inhibited part of the R current in this case. Inset, Representative traces are shown. D, Comparison of inhibitory effects of specific drugs and toxins on the granule cell IBa in α1E KO and control mice is shown. Drugs and toxins were used in the following concentrations: nimodipine (Nimo), 500 nm; ω-conotoxin GVIA (CTx), 500 nm; ω-Aga IVA (Aga), 200 nm; SNX-482 (SNX), 1 μm; Cd2+, 100 μm. The data from the two parental control mouse strains were pooled. SNX-482 was only effective in inhibiting the IBa from control mice (**p < 0.01). The numbers inparentheses represent the number of experiments.E, The effects of the specific drugs and toxins on theIBa from the two control mouse strains did not differ significantly from each other. The numbers inparentheses represent the number of experiments.Resist, Resistant; wt, wild type.
- Fig. 4.
Effects of drugs and toxins on theIBa in DRG neurons from wild-type and α1E KO mice. A, Plot of the peakIBa versus time in a DRG neuron from an α1E KO mouse. An R current still remained after sequential application of the different drugs and toxins. TheIBa from α1E KO mice was insensitive to SNX-482. Inset, Representative currents.B, Time course of the peakIBa from a wild-type DRG neuron exhibiting sensitivity to SNX-482. C, Time course of the peakIBa from a wild-type DRG neuron that was insensitive to SNX-482. D, Average inhibition (mean ± SEM) of the peak IBa by specific drugs and toxins. Drugs and toxins were used in the following concentrations: nimodipine, 2 μm; ω-conotoxin GVIA, 1 μm; ω-Aga IVA, 200 nm; SNX-482, 1 μm. DRG neurons from α1E KO mice (n = 7) and a population of DRG neurons from wild-type mice (n= 8) were insensitive to SNX-482 application. TheIBa inhibition by SNX-482 in sensitive DRG neurons (n = 10) was significant (p < 0.01). E, Interrelationship between sensitivity to ω-Aga IVA and SNX-482 in wild-type DRG neurons (n = 8). The correlation was significant (p = 0.0265) using the Spearman rank correlation test.
Tables
KO Wild type Total current 364.9 ± 50.4 375.8 ± 47.6 Nimodipine 36.1 ± 8.2 44.7 ± 8.4 ω-Aga IVA 75.0 ± 15.0 105.3 ± 15.6 ω-CTx GVIA 139.2 ± 30.7 95.4 ± 22.1 SNX-482 1.8 ± 1.3 45.5 ± 6.9 Resistant 112.8 ± 26.8 84.9 ± 20.54 Comparison of the peak IBa (pA) from cultured mouse cerebellar granule cells (α1E knock-out,n = 5; wild type, n = 9). Data represent peak current amplitudes (pA) blocked by the specific toxin or drug indicated and the remaining resistant current. Data are given as mean ± SEM.
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