Abstract
Glucose-stimulated insulin secretion (GSIS) relies on β-cell Ca2+ influx, which is modulated by the two-pore-domain K+ (K2P) channel, TALK-1. A gain-of-function polymorphism in KCNK16, the gene encoding TALK-1, increases risk for developing type-2 diabetes. While TALK-1 serves an important role in modulating GSIS, the regulatory mechanism(s) that control β-cell TALK-1 channels are unknown. Therefore, we employed a membrane-specific yeast two-hybrid (MYTH) assay to identify TALK-1-interacting proteins in human islets, which will assist in determining signaling modalities that modulate TALK-1 function. Twenty-one proteins from a human islet cDNA library interacted with TALK-1. Some of these interactions increased TALK-1 activity, including intracellular osteopontin (iOPN). Intracellular OPN is highly expressed in β-cells and is upregulated under pre-diabetic conditions to help maintain normal β-cell function; however, the functional role of iOPN in β-cells is poorly understood. We found that iOPN colocalized with TALK-1 in pancreatic sections and coimmunoprecipitated with human islet TALK-1 channels. As human β-cells express two K+ channel-forming variants of TALK-1, regulation of these TALK-1 variants by iOPN was assessed. At physiological voltages iOPN activated TALK-1 transcript variant 3 channels but not TALK-1 transcript variant 2 channels. Activation of TALK-1 channels by iOPN also hyperpolarized resting membrane potential (Vm) in HEK293 cells and in primary mouse β-cells. Intracellular OPN was also knocked down in β-cells to test its effect on β-cell TALK-1 channel activity. Reducing β-cell iOPN significantly decreased TALK-1 K+ currents and increased glucose-stimulated Ca2+ influx. Importantly, iOPN did not affect the function of other K2P channels or alter Ca2+ influx into TALK-1 deficient β-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased β-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced β-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.
Highlights
Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is critical to the maintenance of glucose homeostasis
We identified a number of membrane-associated and cytosolic proteins expressed in pancreatic islets that interact with TWIK-related alkaline pH-activated K2P (TALK)-1 using a powerful technique known as a split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) assay, which has been used to reveal the pancreatic islet glucagon-like peptide 1 receptor (GLP-1R) interactome [23,24,25,26]
To identify TALK-1-interacting proteins (Table 1), a human pancreatic islet cDNA library was generated and screened in a MYTH assay with TALK-1 transcript variant 3 (T3) as a bait
Summary
Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is critical to the maintenance of glucose homeostasis This process requires pancreatic β-cell Ca2+ influx through voltage dependent calcium channels (VDCCs), which are activated by plasma membrane depolarization. The TWIK-related alkaline pH-activated K2P (TALK)-1 channel and the TWIK-related acid-sensitive K2P (TASK)-1 channel hyperpolarize β-cell Vm [4,5,6]. These K2P channels help to set the basal beta-cell Vm with their continuous K+ flux at all physiological Vm [7,8,9]. While K2P channels play an important role in regulating beta-cell Vm and Ca2+ influx, the mechanism(s) controlling beta-cell K2P channels have not been elucidated
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have