Phosphorylation-dependent relief of Rad inhibition of cardiac Caᵥ1.2 channels underlies β-adrenergic increase in heart contraction essential for the fight-or-flight response. Prevailing evidence outline 4 steps involved in PKA-dependent relief of Caᵥ1.2 inhibition by Rad: basally, Rad inhibits Caᵥ1.2 by binding Caᵥβ and the plasma membrane using the G-domain and C-terminus, respectively (step 0), PKA-dependent phosphorylation of Ser residues in Rad C-terminus disengages Rad from the plasma membrane (step 1) and decreases affinity for Caᵥβ (step 2), potentially leading to Rad loss from Caᵥ1.2 nanodomain (step 3).
It is unclear which steps and Rad structural determinants are necessary and sufficient for PKA regulation of CaV channels and the mechanism linking steps 1 and 2 is not entirely understood. Moreover, there is an apparent Rad-concentration-dependence to Caᵥ1.2 regulation wherein PKA activation is unable to overcome over-expressed Rad inhibition of the channel. The basis of this effect is unknown and constitutes a significant gap in our complete understanding of convergent regulation of Caᵥ channels, by Rad and PKA.
We developed a systematic protein engineering-based approach to dissect the distinct steps and determinants involved in PKA modulation of Rad-inhibited Caᵥ channels. Fusing Rad C-terminus to Caᵥβ₃ generated β3-CT which was tethered to the plasma membrane when expressed alone in HEK293 cells and yielded constitutively inhibited channels when co-expressed with CaV2.2. Unexpectedly, PKA activation with forskolin further deepened inhibition of Caᵥ2.2 currents despite being sufficient to release β₃-CT from the plasma membrane. Phosphomimetic mutations in β₃-CT 6SD yielded deeply inhibited Caᵥ2.2 currents that were not further affected by forskolin.
Two CaVβ-binding nanobodies fused to Rad C-terminus, F3-CT and B11-CT, were membrane-targeted yet yielded Caᵥ2.2 currents that were not basally inhibited and decreased by forskolin. Over-expressing wildtype Rad C-terminus (WTCT) by itself with Caᵥ1.2 produced basally inhibited channels that were further reduced by forskolin and co-expression of Caᵥ1.2 with a phosphomimetic Rad C-terminus (CTD) also produced constitutively inhibited channels. Truncated Rad lacking the C-terminus (Rad[1-276]) displayed low affinity to Caᵥβ, discounting a direct role for phosphorylated Rad C-terminus in linking steps 1 and 2. Fusing the protein kinase C C1 domain to Rad[1-276] yielded Rad₂₇₆-C1 which was cytosolic and displayed low affinity to Caᵥβ. Exposure to PdBu recruited Rad₂₇₆-C1 to the plasma membrane, increased affinity for Caᵥβ, and concomitantly inhibited Caᵥ1.2 currents.
These results reveal that all 4 steps are necessary for PKA regulation of Caᵥ channels, membrane association regulates Rad affinity for CaVβ, and the Rad G-domain and C-terminus are replaceable with modular units that mimic their function. Our findings deepen understanding of PKA modulation of Caᵥ channels and provide new insights for developing chemo-genetic Caᵥ channel regulators.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/qg3w-ff06 |
| Date | January 2024 |
| Creators | Gavin, Ariana Cecilia |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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