Abstract
Intracellular chloride concentration ([Cl−]i) in pancreatic β-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl− loaders such as the Na+K+2Cl− co-transporter 1 (Slc12a2) over Cl−extruders of unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and in vitro functional assays, we establish that the “neuron-specific” K+Cl− co-transporter 2 (KCC2, Slc12a5) is expressed in several endocrine cells of the pancreatic islet, including glucagon secreting α-cells, but particularly in insulin-secreting β-cells, where we provide evidence for its role in the insulin secretory response. Three KCC2 splice variants were identified: the formerly described KCC2a and KCC2b along with a novel one lacking exon 25 (KCC2a-S25). This new variant is undetectable in brain or spinal cord, the only and most abundant known sources of KCC2. Inhibition of KCC2 activity in clonal MIN6 β-cells increases basal and glucose-stimulated insulin secretion and Ca2+ uptake in the presence of glibenclamide, an inhibitor of the ATP-dependent potassium (KATP)-channels, thus suggesting a possible mechanism underlying KCC2-dependent insulin release. We propose that the long-time considered “neuron-specific” KCC2 co-transporter is expressed in pancreatic islet β-cells where it modulates Ca2+-dependent insulin secretion.
Highlights
The pancreatic islet is remarkably sensitive to acute changes in plasma glucose levels
The predominant functional presence of NKCC1 relative to other Cl− loaders expressed in rodent β-cells such as Slc12a117, 26, 27, some Slc4a/26a Cl−/HCO3– exchangers[28, 29] or any other potential Cl− extruder of the Slc12a family[30], partially explains the depolarizing driving force of Cl− linked to insulin secretion[3, 19, 20, 31, 32]
Immunoblotting with validated KCC2 antibodies[47] was performed to demonstrate KCC2 protein expression (Fig. 1H) as bands of molecular weights (MW) from ~124 kDa to ~240 kDa, which are expected for KCC2 core/high-mannose (~124 kDa), hybrid-type (130–135 kDa), complex N-glycosylated (~150–160 kDa) and dimers (~240 kDa)[47, 48]
Summary
The pancreatic islet is remarkably sensitive to acute changes in plasma glucose levels. Acute inhibition of NKCC1 with the clinically relevant diuretics BTD or furosemide, inhibits GABAA-mediated plasma membrane depolarization of immature neurons[41], nociceptors[39], chromaffin cells[11] and insulin secretion[5, 16, 17, 27, 31, 42], respectively. These diuretics impair glucose tolerance in mice[27, 43,44,45] and provoke intermittent hyperglycemia in patients treated with these compounds[46]. Our results provide the first indication that KCC2 is expressed in the pancreatic β-cell and plays a role as a modulator of insulin secretion
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