Cav1.3‐selective inhibitors of voltage‐gated L‐type Ca²⁺ channels: Fact or (still) fiction?

Figures

• 

  FIGURE 1. Physiological roles for Cav1.2 and Cav1.3 L‐type Ca2+‐channels (LTCCs). Cav1.2 and Cav1.3 are expressed in most excitable tissues and in most cases even together in the same cells. They can support different physiological functions due to their distinct gating properties (Cav1.3 channels are ‘low‐voltage‐activated LTCCs’), subcellular localization and/or protein‐interactions. Cav1.1 and Cav1.4 show more restricted expression in skeletal muscle and retinal cells and serve key functions for skeletal muscle contraction and photoreceptor signalling, respectively (Zamponi et al., 2015). AV‐node, atrioventricular node; ICa, inward Ca2+ current, IHC, inner hair cells; LTP, long‐term potentiation; OHC, outer hair cells. Adapted from Zamponi et al. (2015), redrawn with permission.

• 

  FIGURE 2. Chemical structures of compounds discussed in this article and L‐type Ca2+‐channel (LTCC) structure. (a) Isradipine (previously also referred to as PN200‐110) is shown as its active (S)‐(+)‐enantiomer, diltiazem as its active (+)‐cis‐diastereomer. Other names that can be found in the literature for cp‐PYT (1‐(3‐chlorophenethyl)‐3‐cyclopentylpyrimidine‐2,4,6‐(1H,3H,5H)‐trione) are compound 8 and BPN‐4689. (b) The structure of the rabbit Cav1.1 channel complex with nifedipine bound in the DHP binding pocket (PDB entry 6JP5 Zhao et al., 2019). α1, β, γ and α2δ1 subunits are coloured pale cyan, pale green, brown and grey, respectively. The DHP blocker nifedipine is shown in dark purple. Lipids facing the DHP binding‐site has been removed for clarity. (c) DHP‐binding site in the Cav1.1 structure with serine residue 1011 (numbering according to rabbit rCav1.1 α1‐sequence, Uniprot P07293) indicated. (d) Homology model of human hCav1.3 α1‐subunit (Uniprot Q01668) based on the rCav1.1 structure, generated with MOE (Molecular Operating Environment, version 2020.09, Molecular Computing Group Inc., Montreal, Canada). The hCav1.3 α1‐subunit (dark green) is shown in comparison to Cav1.1 (pale…

• 

  FIGURE 3. Comparison of the apo‐ and nifedipine‐bound structure of rabbit Cav1.1. The apo structure is shown in violet (PDB entry 5GJV, Wu et al., 2016), whereas the nifedipine‐bound structure (PDB entry 6JP5, Zhao et al., 2019) is depicted in pale cyan. (a) In the nifedipine‐bound Cav1.1 α1‐subunit, the IIIS4‐S5 linker is shifted upwards, whereas helix IIIS5 is rotated towards nifedipine. Red arrows indicate their movements. (b) S1011 of the repeat III pore segment, F1060 and Y1048 of IIIS6 and Q939 of IIIS5 are shown as solid sticks. They all change conformation upon ligand binding as indicated by red arrows. Please note that the S4‐S5 linker and S5 from both Cav1.1 structures are coloured with a darker shade (i.e., nifedipine‐bound Cav1.1 in green cyan, apo‐Cav1.1 in deep purple).

• 

  FIGURE 4. The T1056Y mutation causes a steric clash within the DHP binding site. In the model of hCav1.2 harbouring mutation T1056Y (corresponding to T1066Y in mouse Cav1.2; Sinnegger‐Brauns et al., 2009), the strongly reduced DHP‐sensitivity of the mutant is due to the steric clash between the Y1056 side chain and the DHP‐ring of DHPs (shown for nifedipine, blue), which prevents DHPs from accessing their binding site. This mutation has been used in in vitro and in vivo studies to prevent DHP effects through Cav1.2 channels making existing DHPs ‘selective’ for Cav1.3 in mutant cells. hCav1.2 is shown in grey and Y1056 in IIIS5 is shown as orange sticks. Green sticks represent corresponding T1056 belonging to the wild‐type hCav1.2. The homology model of hCav1.2 is based on PDB entry 6JP5 (Zhao et al., 2019). Model generation and insertion of the mutant residue were conducted with MOE (see legend to Figure 2).

• 

  FIGURE 5. Sequence alignment of rat Cav1.3 α1 subunit constructs used by different groups to study the selectivity of cp‐PYT. Alignments are shown for regions in homologous repeats III and IV with amino acid differences between the constructs. Note that residues M1078 and V1198 (orange) are not different (for details see text). The only difference is alternative splicing of exon 31 (Lieb et al., 2012), which was different in the construct used in the study by Ortner (Ortner et al., 2014) compared to those by the other laboratories (GenBank accession #: AF370009/AF370010; Cooper et al., 2020; Huang et al., 2014; Kang et al., 2012).