2021.7.15_2 Shiqi Tang_thesis.pdf (9.45 MB)

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

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thesis
posted on 22.07.2021, 03:27 by Shiqi TangShiqi Tang

The L-type VGCC subtypes, including subtypes Cav1.1-1.4, 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 Cav1.3 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 Cav1.2, but no selective Cav1.3 inhibitors have been developed yet. We resolved the molecular determinants to differentiate Cav1.2 and Cav1.3 in response to DHP nifedipine. Nifedipine IC50 for Cav1.2 and Cav1.3 are 22nM and 289nM determined by whole-cell patch-clamp. We identified two significant amino acids, Cav1.3/M1030 to Cav1.2/V1036 in the transmembrane IIIS5 and Cav1.3/S1100 Cav1.2/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine.

We found that the Cav1.3/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 V1/2 inactivation with an EC50 of 231nM. P3-1 contains a protein kinase G (PKG) phosphorylation site (RRISE) required for PKG inhibition of Cav1.3 current but not conserved in Cav1.2. We found that the shift in V1/2 inactivation induced by co-expression of Cav1.3 with the Cav1.3/II-III loop/GFP requires the presence of a Cavβ subunit, and Cavβ3 also exhibits selectivity over other β subunits. Significantly, P3-1 shifts the Cav1.2 inactivation to a more positive voltage when co-expressed with either Cavβ2a or Cavβ3, demonstrating the ability of P3-1 to differentiate Cav1.2 and Cav1.3 in a Cavβ-dependent manner.

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 RyR2KO INS-1 cells and generated IRBITKO INS-1 cells. VGCC current density in RyR2KO doubled compared to controls and was also elevated in IRBITKO compared 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.


History

Degree Type

Doctor of Philosophy

Department

Medicinal Chemistry and Molecular Pharmacology

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Gregory Hockerman

Additional Committee Member 2

Val Watts

Additional Committee Member 3

Riyi Shi

Additional Committee Member 4

Yang Yang

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