Characterization of Cav1.4 complexes (α11.4, β2, and α2δ4) in HEK293T cells and in the retina

Abstract

In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 α1, β2, and α2δ4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, and α2δ4 were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Cav1.4 α1 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by β2a and β2X13 variants.

Keywords: Calcium Channel; Calcium-binding Protein; Electrophysiology; Immunohistochemistry; Photoreceptor; Retina.

FIGURE 1.

Cloning and expression of retinal β_2X13 variant. A, schematic of the human CACNB2 gene with exon numbering according to Buraei and Yang (1). Alternatively spliced exons 1, 2, and 7 are linked and denoted by letters. The different domains are indicated: N-terminal (N-term), Src homology 3 (SH3), HOOK, guanylyl kinase (GK), and C-terminal (C-term). GenBank^TM accession numbers are indicated on the right. The distinct peptide sequence encoded by exon 7A in β_2a and exon 7B in β_2X13 is indicated. The zig zag lines indicate the palmitoylation sites at the N terminus. B, qPCR analysis of β_2a and β_2X13 expression in human retina. Values represent mean ΔCt values (Ct values for β_2a and β_2X13 normalized to the Ct values for the internal standard GAPDH) for all cDNA preparations (1–3). Error bars, S.D. (*, p ≤ 0.015, unpaired t test, n = 3). C, RT-PCR analysis of β_2a and β_2X13 expression in human and mouse tissue. For human cDNA, primers were the same as those used in B and Table 1. For mouse cDNA, primers were designed on exon 7A and exon 10 (for β_2a) or on exon 7B and exon 10 (for β_2X13). D, profile of RNA-seq reads from human macula or peripheral retina mapped to exon 6 to exon 8 of the CACNB2 gene. The numbers between parentheses on the right indicate the scale. The location of exon 6 to exon 8 from 18,795.363 to 18,807.311 kb on chromosome 11 is shown at the top.

FIGURE 2.

Alignment of human β_2a and β_2X13 amino acid sequences with mouse β_2a and mouse β_2X13. Cloning of the β_2X13 cDNAs is described in the “Experimental Procedures.” The aligned amino acid sequences are derived from GenBank^TM accession number AF423189.1 for human β_2A, AF465485_1 for human β_2X13, XM_006497320.1 for mouse β2a, and KJ789960 for mouse β_2X13. Similarly to the human β2X13 retinal subunit, the mouse retinal β_2X13 also includes the shorter exon 7B shown in boldface italic letters instead of exon 7A indicated in boldface underlined letters.

FIGURE 3.

Characterization of polyclonal antibodies against β₂ and α₂δ₄. A, immunolabeling of HEK293 cells cotransfected with Ca_v1.4 α₁, β_2X13, and α₂δ₄ (left panels) or untransfected cells (right panels) with rat anti-β₂ or rat anti-α₂δ₄. B, Western blot of lysates of HEK293 cells untransfected (UC) or cotransfected (TC) with Ca_v1.4 α₁, β_2X13 and α₂δ₁, α₂δ₂, α₂δ₃, or α₂δ₄ (left) or cotransfected with Ca_v1.4 α₁, α₂δ₄, and β_1b, β_2X13, β₃, or β₄ (right). Blots were probed with anti-α₂δ₄ or anti-β₂, respectively. C, Western blot of lysates of HEK293 cells untransfected (UC) or cotransfected (TC) with Ca_v1.4 α₁, β_2X13, and α₂δ₄ and probed with anti-β₂, anti-β₂ preadsorbed with specific β_2X13 or nonspecific β_1b (left), or anti-α₂δ₄, or anti-α₂δ₄ preadsorbed with specific α₂δ₄ or nonspecific α₂δ₁ (right). Immunoreactivity was blocked by preadsorption with specific antigen only.

FIGURE 4.

Colocalization of Ca_v1.4 α₁ with β₂ or α₂δ₄ at the photoreceptor synaptic ribbon. Mouse retina sections were double-labeled with anti-Ca_v1.4 α₁ (green) and anti-α₂δ₄ (red) (A) or anti-β₂ (red) (B). The overlay is shown in the right panel together with Hoechst staining. Higher magnification images of the OPL are shown in the bottom panels. Ca_v1.4 shows a high degree of colocalization with β₂ and α₂δ₄, as indicated by the calculated Pearson's correlation coefficient between the Alexa 488-stained Ca_v1.4 and the Alexa 555-stained β₂ (r ≥ 0.710) or α₂δ₄ (r ≥ 0.608). OS, outer segment; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

FIGURE 5.

Isolation of Ca_v1.4 α₁1-β₂-α₂δ₄ complexes from cotransfected HEK293 cells and mouse retina. A and B, Ca_v1.4 α₁ interacts with β_2X13, α₂δ₄, and CaBP4 expressed in HEK293 cells. Coimmunoprecipitations were performed with control mouse IgG or anti-FLAG antibody using lysates of HEK293 cells untransfected (UC) or cotransfected (TC) with β_2X13, α₂δ₄, and CaBP4 with or without (no Ca_v1.4) FLAG-Ca_v1.4 α₁. Experiments were performed in the presence (A) or in the absence of 0.1 mm Ca²⁺ (B). Western blotting (IB) was with anti-FLAG, anti-β₂, anti-α₂δ₄, or anti-CaBP4. Left panels show input lysates. Right panels show coimmunoprecipitated (IP) proteins. Results shown are representative of three experiments. C, Ca_v1.4 α₁ interacts with β₂ and α₂δ₄ in mouse retina. Coimmunoprecipitations were performed using lysates from WT or KO Ca_v1.4 α₁ mouse retina with anti-α₂δ₄ or anti-β₂ antibodies. Anti-Ca_v1.4 α₁ antibody was used for Western blotting. 8% of lysate was used for input.

FIGURE 6.

Ca_v1.4 α₁ interacts with CaBP4, β₂ and α₂δ₄ in mouse retina. Immunohistochemistry (IHC) or PLAs were performed in sections of WT or knock-out mouse retina using anti-Ca_v1.4 α₁ and anti-CaBP4 (A–D), anti-Ca_v1.4 α₁ and anti-β₂ (E–H), anti-Ca_v1.4 α₁ and anti-α₂δ₄ (I–L), anti-Ca_v1.4 (M), anti-β₂ (N), anti-α₂δ₄ (O), no primary antibodies (P), anti-β₂ preadsorbed with β_2a (Q), or anti-α₂δ₄ preadsorbed with α₂δ₄ (R). For IHC, WT mouse retina sections were double-labeled with anti-Ca_v1.4 α₁ (red) and in green anti-CaBP4 (A), anti-Ca_v1.4 α₁ (green) and in red anti-β₂ (E), or anti-α₂δ₄ (I). INL, inner nuclear layer. PLA was performed on retina from WT (B, F, J, and M–R), CaBP4 KO (C), or Ca_v1.4 KO (G and K) mice. Sites of protein interaction are visualized as red spots. Quantification of PLA spots are shown as mean ± S.E. (error bars) for Ca_v1.4/CaBP4 in WT (n = 7) versus CaBP4 KO mice (n = 3) (p < 0.001; Student's t test) (D), for Ca_v1.4-β₂ in WT (n = 7) versus Ca_v1.4 KO mice (n = 3) (p < 0.001) (H), and for Ca_v1.4-α2δ4 in WT (n = 6) versus Ca_v1.4 KO mice (n = 3) (p < 0.001) (L). For all images, nuclei are labeled with Hoechst. Scale bar, 5 μm.

FIGURE 7.

Differential modulation of Ca_v1.4 properties by α₂δ₁ and α₂δ₄. A and B, Ba²⁺ currents (I_Ba) were evoked by 50-ms depolarizations from a holding voltage of −80 mV in HEK293T cells transfected with Ca_v1.4 containing α₂δ₁ (n = 8) or α₂δ₄ (n = 9) and β_2X13. A, representative I_Ba traces during a 50-ms depolarization to −70, 0, and +30 mV. B, current-voltage (I-V) plots for data obtained as in A. C, tail currents were evoked after 20-ms depolarization to various voltages from a holding voltage of −80 mV and repolarization to −60 mV. Tail currents were normalized to that obtained after a +80-mV pulse and plotted against test voltage. D, inactivation of I_Ba evoked by 5-s pulses from −80 to 0 mV. Left, representative traces. Right, inactivation was measured by dividing residual current amplitude (I_res) by the peak amplitude (I_peak) in cells transfected with Ca_v1.4 containing α₂δ₁ (n = 5) or α₂δ₄ (n = 6), p = 0.59, by Student's t test. The time constant, τ, was obtained by fitting I_Ba decay with a single exponential function and was not different for α₂δ₁ and α₂δ₄; p = 0.88, Student's t test. Error bars, S.E.

FIGURE 8.

Differential modulation of Ca_v1.4 properties by β_2a and β_2X13. A–C, same as in Fig. 7 except for Ca_v1.4 containing β_2a (n = 10) or β_2X13 (n = 10) and α₂δ₄. D, same as in Fig. 7D except for Ca_v1.4 containing β_2a (n = 7) or β_2X13 (n = 7), and I_Ba was evoked by either 5- or 10-s pulses from −80 to +10 mV. Left, representative traces are evoked by 10-s pulses. Right, I_res/I_peak (*, p = 0.01 for 5-s pulses; p = 0.04 for 10-s pulses, by Student's t test) and τ (p = 0.32 for 5-s pulses, whereas p = 0.16 for 10-s pulses by Student's t test). Error bars, S.E.

FIGURE 9.

Unlike their distinct effects on Ca_v1.4, β_2a and β_2X13 do not differentially modulate Ca_v1.2 inactivation. A–C, same as in Fig. 7 except for Ca_v1.2 channels containing β_2a (n = 10) or β_2X13 (n = 10) and α₂δ₄. D, same as in Fig. 7D except for Ca_v1.2 containing β_2a (n = 9) or β_2X13 (n = 9), and I_Ba was evoked by 5-s pulses from −80 to 0 mV. Left, representative traces are evoked by 5-s pulses. Right, I_res/I_peak (*, p = 0.75, by Student's t test) and τ (p = 0.81 by Student's t test). Error bars, S.E.