Differentiation of Cav1.2 and Cav1.3 pharmacology and role of RyR2 in pancreatic beta-cell electrophysiology

<p></p><p>The<b> </b>L-type VGCC subtypes, including subtypes Ca_v1.1-1.4, 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_v1.3 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_v1.2, but no selective Ca_v1.3 inhibitors have been developed yet. We resolved the molecular determinants to differentiate Ca_v1.2 and Ca_v1.3 in response to DHP nifedipine. Nifedipine IC₅₀ for Ca_v1.2 and Ca_v1.3 are 22nM and 289nM determined by whole-cell patch-clamp. We identified two significant amino acids, Ca_v1.3/M1030 to Ca_v1.2/V1036 in the transmembrane IIIS5 and Ca_v1.3/S1100 Ca_v1.2/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine. </p> <p>We found that the Ca_v1.3/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_1/2 inactivation with an EC₅₀ of 231nM. P3-1 contains a protein kinase G (PKG) phosphorylation site (RRISE) required for PKG inhibition of Ca_v1.3 current but not conserved in Ca_v1.2. We found that the shift in V_1/2 inactivation induced by co-expression of Ca_v1.3 with the Ca_v1.3/II-III loop/GFP requires the presence of a Ca_vβ subunit, and Ca_vβ₃ also exhibits selectivity over other β subunits. Significantly, P3-1 shifts the Ca_v1.2 inactivation to a more positive voltage when co-expressed with either Ca_vβ_2aor Ca_vβ₃, demonstrating the ability of P3-1 to differentiate Ca_v1.2 and Ca_v1.3 in a Ca_vβ-dependent manner.</p> <p><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) Ca²⁺ 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 IP₃ receptor binding protein (IRBIT) in RyR2^KO INS-1 cells and generated IRBIT^KO INS-1 cells. VGCC current density in RyR2^KO doubled compared to controls and was also elevated in IRBIT^KOcompared to control cells. All HVA Ca²⁺ 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. </p><br><p></p>