The Molten Globule State of Maltose Binding Protein: Structural Characterization by Epr Spectroscopy

Abstract

Mouse islets lacking TALK-1 (KO) exhibit accelerated Ca 2þ c oscillations and enhanced insulin secretion relative to controls (WT). The Ca 2þ c oscillation frequency is affected by K slow , a repolarizing Ca 2þ -dependent K þ current. K slow is reduced in KO b-cells, possibly contributing to the accelerated Ca 2þ c oscillations of KO islets. Since TALK-1 activity is not regulated by Ca 2þ c , we assessed whether TALK-1 impacts Ca 2þ release from endoplasmic reticulum (ER) stores (Ca 2þ ER ), which modulates K slow . We find lower basal Ca 2þ c in KO b-cells, and application of the SERCA inhibitor CPA causes a greater increase in Ca 2þ c in KO versus WT b-cells. These observations suggest that TALK-1 regulates Ca 2þ ER . Therefore, we tested how heterologous expression of TALK-1 in HEK293 cells affects Ca 2þ ER . TALK-1 overexpression significantly increases basal Ca 2þ c and reduces the CPA-induced Ca 2þ c response. However, expression of the K2P channels TREK-1 or TREK-2 does not affect Ca 2þ ER . Furthermore, K þ -impermeable mutant TALK-1 channels do not recapitulate Ca 2þ ER defects, thus, TALK-1 K þ channel function presumably modulates Ca 2þ ER . Consistent with an intracellular role for TALK-1, fluorescence microscopy reveals expression of TALK-1 in the b-cell ER. Additionally, nuclear patch-clamp experiments demonstrate the presence of functional TALK-1 channels on the outer nuclear membrane, which is continuous with the ER. Moreover, specific inhibition of endogenous TALK-1 channels with expression of a K þ -impermeable mutant increased human b-cell Ca 2þER . Together, our data suggest that TALK-1 channel activity augments islet-cell Ca 2þ ER ''leak.'' Altered TALK-1 channel function may perturb islet-cell Ca 2þ ER , which can affect Ca 2þ c handling, hormone release, and contribute to disease pathogenesis.

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy

“…In the vicinity of the active site (MBP 07–11), the structures hardly differ between the MG and native state as shown by EPR spectroscopy. These findings corroborate previous findings that MBP can still bind maltose in the MG state and confirm the assumption that a defined structure of MBP exists near the binding pocket even in the MG state. , …”