Differentiation of CAV1.2 and CAV1.3 Pharmacology and Role of RYR2 in Pancreatic Beta-Cell Electrophysiology

Shiqi Tang, Purdue University

Abstract

The L-type VGCC subtypes, including subtypes Cav1-1.4, 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 found that this 12-fold difference in potency was largely accounted for by amino acid sequence divergence within the DHP binding pocket originally mapped in Cav1.2. Specifically, we identified two significant amino acids, Cav1.3/M1030 toCav1.2/V1036 in the transmembrane IIIS5 and Cav1.3/S1100 Cav12/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine. We generated aCav1.3 mutant channel containing both the M/V switch in IIIS5 and the IIIS5-3P loop of Cav1.2, which was blocked by nifedipine with an IC50 of 42nM. Furthermore, we found that switching amino acids at positions F1052, Q1089, D1092, N1094, and S100 within the Cav1.3 IIIS-3P loop to the corresponding amino acids in Cav1.2, reduced the EC50 of the L-type channel agonist FPL 64176 from 854nM to 133nM, essentially close to that of Cav1.2 at 103nM. Developing L-VGCC subtype-selective inhibitors based on existing small molecules has been challenging. Therefore, we asked if the intracellular loops of Cav1.2 and Cav1.3, which are highly divergent, could be targeted for selective modulation of these two subtypes. First, 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. Next, we expressed Cav1.3/β3/α2δ1 in tsA-201 cells and found that overexpression of the Cav1.3 II-III loop selectively shifts Cav1.3 inactivation by - 15mV, but not Cav1.2. To refine the significant residues in the Cav1.3 II-III loop, we created GFP fusions with the N and C-terminal (NT and CT) half of the Cav1.3/II-III loop co-expressed with α21 and β3 and found that both could shift the inactivation of Cav1.3 to hyperpolarizing potentials. We introduced several synthetic peptides, and peptide P3-1 from CT 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.

Degree

Ph.D.

Advisors

Hockerman, Purdue University.

Subject Area

Pharmacology|Physiology|Aging|Disability studies|Endocrinology|Medicine|Mental health|Neurosciences|Pharmaceutical sciences|Public health

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