Abstract
The ATP-sensitive potassium channel (KATP) controls insulin release in pancreatic beta-cells and also modulates important functions in other cell types. In this study we report that anionic phospholipids activated KATP in pancreatic beta-cells, cardiac myocytes, skeletal muscle cells, and a cloned KATP composed of two subunits (SUR/Kir6. 2) stably expressed in a mammalian cell line. The effectiveness was proportional to the number of negative charges on the head group of the anionic phospholipid. Screening negative charges with polyvalent cations antagonized the effect. Enzymatic treatment with phospholipases that reduced charge on the lipids also reduced or eliminated the effect. These results suggest that intact phospholipids with negative charges are the critical requirement for activation of KATP, in distinction from the usual cell signaling pathway through phospholipids that requires cleavage. Mutations of two positively charged amino acid residues at the C terminus of Kir6. 2 accelerated loss of channel activity and reduced the activating effects of phospholipids, suggesting involvement of this region in the activation. Metabolism of anionic phospholipids in plasmalemmal membrane may be a novel and general mechanism for regulation of KATP and perhaps other ion channels in the family of inward rectifiers.
Highlights
The ATP-sensitive potassium channel (KATP)1 is a highly regulated channel type important in the physiology and pathophysiology of pancreas, heart, vascular smooth muscle, and perhaps other tissues as well [1,2,3,4,5]
Currents of native KATPs were recorded from inside-out patches of pancreatic -cells (Fig. 1A), cardiac ventricular myocytes (Fig. 1B), and sarcomeric membrane vesicles from skeletal muscle cells (Fig. 1C)
KATP current expressed in HEK cells stably transfected by cloned cDNA of rat SUR (rSUR) and rKir6.2 was studied (Fig. 1D)
Summary
Cell Isolations—Single pancreatic -cells were enzymatically (0.05% trypsin) dispersed from isolated islets of Wistar rats collected by centrifugation through a discontinuous gradient of Ficoll [17]. Solutions and Chemicals—The pipette solution (extracellular side, 140-Kϩ-pipette) contained (in mM) KCl (140.0), CaCl2 (1.8), MgCl2 (0.5), HEPES (5.0), and glucose (5.5), pH 7.4. The pipette solution for membrane vesicles from skeletal muscle cells and cloned KATP (SUR/Kir6.2) contained (in mM) NaCl (140.0), CaCl2 (1.8), MgCl2 (0.5), KCl (10.0), HEPES (5.0), and glucose (5.5). The bath solution (cytoplasmic side, 140-Kϩ-bath) contained (in mM) KCl (142.0), HEPES (5.0), glucose (5.5), and EGTA (2.0), pH 7.4. For whole cell clamp experiments, the pipette solution (140-Kϩ with Mg-ATP) contained (in mM) KCl (140.0), EGTA (2.0), HEPES (5.0), glucose (5.5), and Mg-ATP (5.0), pH 7.3. The bath solution (5.4-Tyrode solution) contained (in mM) KCl (5.4), NaCl (137.0), CaCl2 (1.8), MgCl2 (0.5), and HEPES (5.0), and the pH was adjusted to 7.4. Anionic phospholipids were dispersed in the solution with 30-min sonication Data were acquired by digitizing at 2 kHz and analyzed by a pClamp 6.0 (Axon Instruments) and graphic plotting software running on a PC-compatible computer (Gateway 2000)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
