ATP modulates interaction of syntaxin-1A with sulfonylurea receptor 1 to regulate pancreatic beta-cell KATP channels

Abstract

ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet β-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic β-cells demonstrated that reduced ATP concentrations increased K(ATP) channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K(ATP) channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit β-cell K(ATP) channel gating, they could also influence the sensitivity of K(ATP) channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic β-cell K(ATP) channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.

FIGURE 1.

ATP dose-dependently inhibited Syn-1A binding to SUR1. GST-Syn-1A bound to glutathione-agarose beads was used to pull down SUR1 from SUR1/Kir6.2 expressing HEK293 cell lysate extract in the presence of increasing concentrations of ATP along with (A) or without (B) 2 mm MgCl₂. The top panels in A and B show representative blots, and the bottom panels are the summaries of quantitative densitometry scanning of the specific bands. Free [ATP], free [Mg2+], and [MgATP] were calculated and shown on the top of the representative blot in B. The value from the assay in the absence of ATP and MgCl₂ was considered as 100% (control). The results are expressed as mean ± S.E. (n = 3). The arrows in B point to the ATP concentrations (0.5 and 2 mm, respectively) which were used in the FRET study (Fig. 2).

FIGURE 2.

ATP inhibited Syn-1A interaction with SUR1 in living cells. Shown in A (ia), A (iia), and A (iiia) are representative recordings of the FRET signals on the membrane of a same HEK cell expressing Syn-1A-mCherry, SUR1-EGFP, and Kir6.2 prior to (A, ia) and after addition of 0.5 (A, iia) and then 2 mm ATP (A, iiia), each for 3 min. The excitation wavelength was 488 nm, and fluorescence emission was measured at 605–655 nm. The arrows indicate the area where FRET signals decreased significantly after applying ATP. The bar indicates 5 μm. A (ib), A (iib), and A (iiib), the FRET efficiency in the same cell corresponding to A (ia), A (iia), and A (iiia), respectively. The vertical scale bar indicates the FRET efficiency in pseudocolor. A (iva) and A (ivb), FRET signal recording and efficiency in control cells expressing EGFP and mCherry. B, summary of FRET efficiency before and after addition of 0.5 or 2 mm ATP. ***, p < 0.001 compared with 0 ATP (n = 12). NS, no significant difference.

FIGURE 3.

Syn-1A inhibited rat islet β-cell K_ATP currents in the presence of 0.3 but not 1 mm intracellular ATP. Cells were treated with 1 μm Syn-1A or 1 μm GST under whole-cell patch clamp recording mode. A, time course of K_ATP currents recorded in the presence of 0.3 mm intracellular MgATP. K_ATP currents were confirmed by application of 300 μm tolbutamide as indicated by arrows. B, time course of K_ATP currents in the presence of 1.0 mm intracellular MgATP. C, summary of the results. The maximum current magnitude was normalized with cell size to yield current density (pA/pF). *, significant difference compared with the control (p < 0.05). Results are expressed as mean ± S.E. (n = 6–11).

FIGURE 4.

K_ATP channel sensitivity to Syn-1A inhibition is enhanced at lower but not at higher ATP concentration in endogenous Syn-1A-depleted cells. A, Western blotting analysis of Syn-1A expression in HEK293 cells transfected with Syn-1A or co-transfected with Syn-1A plus BoNT/C1 (Ai), and in INS-1E cells transfected with BoNT/C1 (Aii), as indicated. The cells were transfected with empty vector as a control. B–D, electrophysiological study. Effects of Syn-1A (0.3 μm) on K_ATP channels at various concentrations of ATP were examined. 0.3 μm GST was used as a negative control. Bi and Di, representative traces obtained from INS-1E cells transfected with EGFP alone. Ci and Dii, representative traces from INS-1E cells co-transfected with BoNT/C1 and EGFP. Bii and Cii, summary of results presented as means ± S.E. (n = 4). The differences (Δ) in current amplitude between application of GST and Syn-1A were calculated as a percentage of the maximal current (I_max) of each individual patch. *, p < 0.05.