Differentiation of Cav1.2 and Cav1.3 pharmacology and role of RyR2 in pancreatic beta-cell electrophysiology
Creators: name: Tang, Shiqi
Publisher: name: Purdue University Graduate School
Year: 2021
Subjects: Pharmacology and pharmaceutical sciences not elsewhere classified

description: The<b> </b>L-type VGCC subtypes, including subtypes Ca<sub>v</sub>1.1-1.4, have been shown to play critical roles in various cellular activities, including muscle contraction, hormone secretion, and neurotransmitter release. Recent research indicates the potential involvement of Ca<sub>v</sub>1.3 in various neurological and psychiatric disorders, such as the early onset of Parkinson’s disease and substance abuse disorders. Non-selective L-VGCC subtype blockers such as dihydropyridines (DHPs) are used to treat hypertension and angina because they potently inhibit Ca<sub>v</sub>1.2, but no selective Ca<sub>v</sub>1.3 inhibitors have been developed yet. We resolved the molecular determinants to differentiate Ca<sub>v</sub>1.2 and Ca<sub>v</sub>1.3 in response to DHP nifedipine. Nifedipine IC<sub>50</sub> for Ca<sub>v</sub>1.2 and Ca<sub>v</sub>1.3 are 22nM and 289nM determined by whole-cell patch-clamp. We identified two significant amino acids, Ca<sub>v</sub>1.3/M1030 to Ca<sub>v</sub>1.2/V1036 in the transmembrane IIIS5 and Ca<sub>v</sub>1.3/S1100 Ca<sub>v</sub>1.2/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine. We found that the Ca<sub>v</sub>1.3/II-III loop fused to eGFP decreased glucose-activated action potential (GSAP) frequency by ~80% in the pancreatic β-cell. We introduced several synthetic peptides, and peptide P3-1 from C-terminal induced a -16mV shift in V<sub>1/2</sub> inactivation with an EC<sub>50</sub> of 231nM. P3-1 contains a protein kinase G (PKG) phosphorylation site (RRISE) required for PKG inhibition of Ca<sub>v</sub>1.3 current but not conserved in Ca<sub>v</sub>1.2. We found that the shift in V<sub>1/2</sub> inactivation induced by co-expression of Ca<sub>v</sub>1.3 with the Ca<sub>v</sub>1.3/II-III loop/GFP requires the presence of a Ca<sub>v</sub>β subunit, and Ca<sub>v</sub>β<sub>3</sub> also exhibits selectivity over other β subunits. Significantly, P3-1 shifts the Ca<sub>v</sub>1.2 inactivation to a more positive voltage when co-expressed with either Ca<sub>v</sub>β<sub>2a </sub>or Ca<sub>v</sub>β<sub>3</sub>, demonstrating the ability of P3-1 to differentiate Ca<sub>v</sub>1.2 and Ca<sub>v</sub>1.3 in a Ca<sub>v</sub>β-dependent manner. <b> </b>Failure of pancreatic β-cells to secrete enough insulin to maintain glucose homeostasis is a hallmark of Type 2 diabetes. However, the consequences of the dysregulation of the endoplasmic reticulum (ER) Ca<sup>2+</sup> channel ryanodine receptor-2 (RyR2) in pancreatic β-cells are not fully understood. Therefore, we characterized the electrical activity in INS-1 in which RyR2 has been deleted via CRISPR/Cas9 gene editing. We observed a decreased level of IP<sub>3</sub> receptor binding protein (IRBIT) in RyR2<sup>KO</sup> INS-1 cells and generated IRBIT<sup>KO</sup> INS-1 cells. VGCC current density in RyR2<sup>KO</sup> doubled compared to controls and was also elevated in IRBIT<sup>KO </sup>compared to control cells. All HVA Ca<sup>2+</sup> channels were upregulated, determined by fractional current blocked by nifedipine. We also found that GSAP frequency is doubled by RyR2 deletion due to failure to activate apamin sensitive SK (small conductance calcium-activated potassium) channels. <br>