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Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

ABSTRACT: Lithium (Li⁺) is the first-line therapy for bipolar disorder and a candidate drug for various diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke. Despite being the captivating subject of many studies, the mechanism of lithium's therapeutic action remains unclear. To date, it has been shown that Li⁺ competes with Mg²⁺ and Na⁺ to normalize the activity of inositol and neurotransmitter-related signaling proteins, respectively. Furthermore, Li⁺ may co-bind with Mg²⁺-loaded adenosine or guanosine triphosphate to alter the complex's susceptibility to hydrolysis and mediate cellular signaling. Bipolar disorder patients exhibit abnormally high cytosolic Ca²⁺ levels and protein kinase C (PKC) hyperactivity that can be downregulated by long-term Li⁺ treatment. However, the possibility that monovalent Li⁺ could displace the bulkier divalent Ca²⁺ and inhibit PKC activity has not been considered. Here, using density functional theory calculations combined with continuum dielectric methods, we show that Li⁺ may displace the native dication from the positively charged trinuclear site in the C2 domain of cytosolic PKCα/γ. This would affect the membrane-docking ability of cytosolic PKCα/γ and reduce the abnormally high membrane-associated active PKCα/γ levels, thus downregulating the PKC hyperactivity found in bipolar patients.

SUBMITTER: Grauffel C

PROVIDER: S-EPMC8359144 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.

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Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

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