<p></p><p>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. </p>
<p>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.</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) 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. </p><br><p></p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14984691 |
| Date | 22 July 2021 |
| Creators | Shiqi Tang (11134677) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY-NC-SA 4.0 |
| Relation | https://figshare.com/articles/thesis/Differentiation_of_Cav1_2_and_Cav1_3_pharmacology_and_role_of_RyR2_in_pancreatic_beta-cell_electrophysiology/14984691 |
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