The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

Abstract

Background:The mechanisms responsible for Ca V 1.2 regulation by the ␣ 1C C terminus are unknown. Results: Trafficking, basal function, and adrenergic modulation of Ca V 1.2 were not altered in cardiomyocytes of transgenic mice expressing PDZ-deleted ␣ 1C . Conclusion: PDZ-mediated interactions are not required for Ca V 1.2 trafficking and function in the heart. Significance: The regulation of Ca V 1.2 by auxiliary proteins does not depend on the PDZ ligand motif in the heart.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics

“…Male and female mixed-strain mice, 6-weeks to 4-months of age were used. Sample sizes exceeded the number of samples determined by power calculations, which were based on effect sizes previously reported 16,17,42,43 . Number of animals was always greater than 3 per genotype.…”

“…Mice ventricular myocytes were isolated by enzymatic digestion using a Langendorff perfusion apparatus as previously described 16,17,42,43,60 . Cardiomyocytes were isolated from 8-12 week-old non-transgenic and transgenic mice.…”

Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+Channels at Excitatory Synapses

“…This effect of densin may be particularly important to replenish Ca v 1.2 channels endocytosed during high levels of neuronal excitation (Green et al, 2007;. The NT of Ca v 1.2 contains binding sites for multiple proteins including calmodulin (Ivanina et al, 2000), CaMKII (Simms et al, 2015), and CaBP1 (Zhou et al, 2005). Therefore, the loss of Ca v 1.2-HA-⌬NT interactions with densin and potentially other proteins could have collectively contributed to the complete absence of cell surface labeling of these mutant channels in dendrites (Fig.…”

“…For example, the PDZ domains of Erbin and Shank interact with the C termini of Ca v 1.3, but not Ca v 1.2 Calin-Jageman et al, 2007). Although deletion of the Ca v 1.2 PDZ-binding site does not affect Ca v 1.2 trafficking in neurons or cardiac myocytes (Yang et al, 2015), deletions within the NT domain increase the cell surface density and maximal open probability of Ca v 1.2 in Xenopus oocytes (Wei et al, 1996;Kanevsky and Dascal, 2006). Binding of densin to the NT domain may mimic the effect of these deletions because densin not only increases the number of functional channels, but also their relative open probability (Fig.…”

“…For cell transfection, cDNAs corresponding to Ca v 1.2 subunits [rbcII (GenBank #M67515), ␤ 2a (GenBank #NC013684) and ␣ 2 ␦ 1 (GenBank #M76559.1)] were expressed from the pcDNA3.1ϩ vector (Invitrogen). The following constructs were described previously: Ca v 1.2/pcDNA3.1ϩ (FLAG-tagged), Ca v 1.2⌬NT/pcDNA3.1ϩ (FLAGtagged) lacking aa 1-109, GST-tagged Ca v 1.2 intracellular domains: NT (aa 1-124); loop I-II (aa 406 -524); loop II-III (aa 754 -902); loop III-IV (aa1170 -1222); CT (aa 1942CT (aa -2144 (Zhou et al, 2004;Zhou et al, 2005); GST-tagged C-terminal domain of Ca v 1.3 (aa 1962-2155) (CalinJageman et al, 2007); GFP-tagged densin (GenBank #NM_057142.1 (Jiao et al, 2008)); p␤A-eGFP and p␤A-Ca v 1.2-hemagglutinin (HA) (Altier et al, 2002;Obermair et al, 2004). GST-tagged fragments of Ca v 1.2 NT were generated by PCR amplification and subcloning into BamHI and EcoRI sites of pGEX-4T1.…”