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. 2004 May 15;113(10):1430–1439. doi: 10.1172/JCI20208

Figure 1.

Figure 1

Construction of Cav1.2DHP–/– mice. (A) Map of WT (a) and mutant (b) Cav1.2 α1 alleles. neo, neomycin resistance gene flanked by loxP elements (triangles); 24, exon containing T1066Y mutation in IIIS5. Primers P1 to P5 were used to detect T1066Y. (c) Mutated allele after neo excision. Bam, BamHI; Csp, Csp45I; S, SpeI. (B) Southern blot analysis of genomic DNA from WT, heterozygous (Het), and homozygous (Cav1.2DHP–/–) mutants (Mut) after BamHI digestion using the probe indicated in A. Arrows indicate bands from WT (7.5 kb) and mutant alleles (9.5 kb). (C) Immunoblots (see Methods): Arrows indicate migration of short (160–190 kDa) and long (220 kDa) forms of specific Cav1.2 α1 immunoreactivity. Migration of molecular mass standards (kDa) is indicated. One representative experiment of three is shown. (D) Equilibrium (+)-[3H]isradipine binding (0.9–1.4 nM) to heart or brain membranes (10–20 ∝g/ml protein). Specific binding in heterozygous and homozygous brain is expressed as percentages of WT binding. Nonspecific binding was defined in the presence of 1 ∝M isradipine. (E) (+)-[3H]isradipine saturation binding analysis in brain membranes of WT (closed circles), heterozygous (open squares), and homozygous mutant (open circles) mice. One representative experiment of five is shown. WT, KD = 0.076 nM, maximal binding capacity (Bmax) = 23.1 pM (462 fmol/mg); heterozygous mutants: KD = 0.067 nM, Bmax = 12.8 pM (255 fmol/mg); homozygous mutants: KD = 0.347 nM, Bmax = 3.81 pM (76 fmol/mg).