Abstract
In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.
Highlights
A tight control of the insulin concentration in the blood is essential to maintain blood sugar levels within a narrow range and to prevent diabetes mellitus in humans. β-cells of the pancreas are unique with regard to their production and release of insulin
Cells were pre-incubated for 30 min in CX-4945 or DMSO. (B) Fura-2 Ca2+ imaging experiments with HEKα2 cells transfected with cDNA encoding encoding fusion p hanced green fluorescent protein (EGFP)–CK2α fusion proteins and stimulated with pregnenolone sulfate
Bated for 30 min in CX‐4945 or DMSO. (B) Fura‐2 Ca imaging exp transfected with cDNA encoding EGFP–CK2α fusion proteins and sulfate (PS)/CIM0216
Summary
A tight control of the insulin concentration in the blood is essential to maintain blood sugar levels within a narrow range and to prevent diabetes mellitus in humans. β-cells of the pancreas are unique with regard to their production and release of insulin. The main stimulus for the fusion of insulin-containing granules with the β-cell membrane and the release of insulin is the elevation of the Ca2+ concentration within the cytosol of the cell ([Ca2+ ]cyt ) [1,2]. This increase is determined by a number of pathways including Ca2+. Entry into the cell across the plasma membrane [2] Among these pathways, the opening of voltage-gated Ca2+ channels (Cav) is regarded as the main mechanism for the rise of [Ca2+ ]cyt [1,2]. Ca signals of green fluorescent cells (green) cells (black) of the same dish as well as to non‐transfected cells
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