Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain

Abstract

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different alpha1 and four different beta subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The beta subunit-specific antibodies identified the presence of beta1a in skeletal muscle; beta2 in heart; and beta2, beta3, and beta4 in brain. The beta3 protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The beta4 protein was also widely expressed, most prominently in the cerebellum. beta2 protein was expressed at only low levels. In situ hybridization with beta subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the alpha1A, alpha1B, alpha1C, and alpha1D subunits showed a broad distribution of alpha1A and alpha1B transcripts, whereas the expression level of alpha1C and alpha1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that beta4 may associate predominantly, but probably not exclusively, with the alpha1A subunit, and beta3 with the alpha1B subunit. In certain brain areas such as the habenula, the beta3 subunit may associate with other alpha1 subunits too. Furthermore, the beta2 subunit may form complexes with different alpha1 subunits in brain and cardiac muscle. These results demonstrate that a given beta subunit may associate with different alpha1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Fig. 2.

Controls for antibody specificity, histoblots, and ISH sections. A, Microsomal membranes from HEK 293 cells transfected with control vector (lane 1) or full-length β₃ cDNA expression vector (lanes 2,3) were electrophoresed on a 7.5% SDS gel and blotted onto nitrocellulose. Lanes 1 and 2 were probed with the anti-β₃ antibody preincubated in the absence (−) and lane 3 with the anti-β₃antibody preincubated in the presence (+) of 10 μmpeptide B30. B, A sagittal histoblot section adjacent to the section of Figure 3B was probed with the anti-β₂-specific antibody blocked with 10 μm peptide B36. C, A sagittal ISH section adjacent to the section of Figure 4B was labeled with the β₂ sense riboprobe.

Fig. 1.

Expression of β subunit proteins in rat tissues. Microsomal membranes (50 μg per lane) of rat liver (Li), skeletal muscle (Sk), brain (Br), heart (He), and aorta (Ao) were separated using 7.5% SDS-PAGE and transferred to nitrocellulose. The blots were probed with the anti-β_common antibody (A), the anti-β₂ specific antibody (B), the anti-β₃ specific antibody (C), and the anti-β₄ specific antibody (D). In (B), the lane containing the separated brain proteins (Br) was from a separate immunoblot and was exposed four times longer than the other lanes. Molecular mass standards (× 10⁻³) are indicated on the left of each blot.

Fig. 3.

Histoblots showing the neuronal distribution of β subunit proteins. Sagittal sections of rat brain were transferred to nitrocellulose and labeled with the anti-β_commonantibody (A), the anti-β₂ antibody (B), the anti-β₃ antibody (C), and the anti-β₄ antibody (D).

Fig. 5.

Distribution of β subunits in rat brain.A, Histoblot of a coronal section labeled with the β₃-specific antibody. B, ISH of an adjacent coronal section with the β₃-specific riboprobe.C, Histoblot of an adjacent coronal section labeled with the β₄-specific antibody. D, ISH of a horizontal section with the riboprobe directed against β₄mRNA.

Fig. 4.

Neuronal distribution of β subunit mRNAs. Autoradiographic film images of sagittal sections hybridized with antisense riboprobes specific for β₁ subunit mRNA (A), β₂ subunit mRNA (B), β₃ subunit mRNA (C), and β₄subunit mRNA (D).

Fig. 6.

Cellular localization of β subunit mRNA in the cerebellum, olfactory bulb, and cortex. Dark-field microscopy views showing emulsion-dipped sagittal sections through the cerebellum labeled with antisense riboprobes specific for β₁ subunit mRNA (A), β₂ subunit mRNA (B), β₃ subunit mRNA (C), and β₄ subunit mRNA (D). M, Molecular cell layer; P, Purkinje cell layer;G, granular cell layer.E, F, Coronal sections through the olfactory bulb hybridized with the riboprobes directed against the β₃ subunit mRNA (E) and β₄ subunit mRNA (F).Gr, Granular layer; Mi, mitral cell layer; Ep, external plexiform layer; Gl, glomerular layer. G, H, Coronal section through the frontal cortex showing the expression of β₃subunit mRNA (G) and β₄ subunit mRNA.H, I–VI, Cortical layers I–VI. I, Coronal section through the habenular complex labeled with the β₃-specific riboprobe. Magnification, 100×.

Fig. 7.

Neuronal distribution of α₁ subunit mRNAs. Autoradiographic film images of sagittal sections hybridized with antisense riboprobes specific for α_1C subunit mRNA (A), α_1D subunit mRNA (B), α_1B subunit mRNA (C), and α_1A subunit mRNA (D).

Fig. 8.

Cellular localization of the α₁subunit mRNAs in the cerebellum and olfactory bulb.A–D, Sagittal sections through the cerebellum hybridized with riboprobes directed against the α_1Csubunit mRNA (A), α_1D subunit mRNA (B), α_1B subunit mRNA (C) and α_1A subunit mRNA (D).E, F, Sections through the olfactory bulb labeled with the riboprobes specific for α_1D subunit mRNA (E) and α_1A subunit mRNA (F). Abbreviations are as in the legend to Figure6. Magnification, 100×.