Mouse retinal rod bipolar cells express different L-type Ca channel transcripts

Ribbon synapses in the retina provide sustained glutamatergic transmitter release that is controlled by Cav1.4 L-type calcium channels in photoreceptors. In bipolar cells the voltage-gated calcium channels (VGCC) responsible for release are less conclusive. We therefore addressed the VGCCs expressed in mouse rod bipolar cells (RBC). We enriched RBCs by FACS-sorting and quantified mRNA expression levels of Cav1, Cav2 and auxiliary VGCC subunit isoforms, including selected splice variants. Transcript variants of candidate VGCC genes were confirmed in RT-PCR and through screening of RNAseq datasets. The contribution of Cav1.4 to RBC calcium currents was assessed in whole-cell patch-clamp recordings from RBC in retina slices of wild-type (WT) and Cav1.4 knock-out (KO) mouse retinas. In the FACS-sorted RBC we found Cav1.3 in short and long forms, Cav1.4 and a novel splice variant of Cav1.1, accompanied by β2 and α2δ-4. The RNAseq data screening revealed all annotated Cav1.4 exons and splice variants of Cav1.3 with exons 8a, Δ11, 31b and 32. The retinal Cav1.1 transcript is lacking the first 15 exons but contains two novel exons at the N-terminus that get alternatively spliced and which we confirmed by RT-PCR. Though there was substantial transcript of Cav1.4 in RBCs, our whole-cell patch-clamp recordings showed that VGCC currents persist when knocking out Cav1.4 (peak current density WT -4,1 ± 0,94 pA/pF, KO -2,0 ± 0,66 pA/pF, p < 0.0001, Statistical analysis: unpaired students t-test). While Cav1.3 could compensate for Cav1.4, it is unclear whether the retinal Cav1.1 transcript would even form functional channels.