I D Dukes

EXPRESSION AND FUNCTION OF PANCREATIC BETA -CELL DELAYED RECTIFIER K+ CHANNELS : ROLE IN STIMULUS-SECRETION COUPLING

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (βTC3-neo) and β-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in βTC3-neo insulinoma cells and purified rodent pancreatic β-cells was detected and confirmed by immunoblotting in the insulinoma cells. βTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on βTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized β-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis.

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic β-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K channels (K) to insulin secretion via depolarization-driven increases in intracellular Ca ([Ca]). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block K. A membrane-permeant analog, methyl pyruvate, however, produced a block of K, a sustained rise in [Ca], and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of K responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the β-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.

Characterization of a Ca2+ Release-activated Nonselective Cation Current Regulating Membrane Potential and [Ca2+] i Oscillations in Transgenically Derived β-Cells

Although stimulation of insulin secretion by glucose is regulated by coupled oscillations of membrane potential and intracellular Ca2+ ([Ca2+] i ), the membrane events regulating these oscillations are incompletely understood. In the presence of glucose and tetraethylammonium, transgenically derived β-cells (βTC3-neo) exhibit coupled voltage and [Ca2+] i oscillations strikingly similar to those observed in normal islets in response to glucose. Using these cells as a model system, we investigated the membrane conductance underlying these oscillations. Alterations in delayed rectifier or Ca2+-activated K+ channels were excluded as a source of the conductance oscillations, as they are completely blocked by tetraethylammonium. ATP-sensitive K+ channels were also excluded, since the ATP-sensitive K+ channel blocker tolbutamide substituted for glucose in inducing [Ca2+] i oscillations. Thapsigargin, which depletes intracellular Ca2+ stores, and maitotoxin, an activator of nonselective cation channels, both converted the glucose-dependent [Ca2+] i oscillations into a sustained elevation. On the other hand, both SKF 96365, a blocker of Ca2+ store-operated channels, and external Na+ removal suppressed the glucose-stimulated [Ca2+] i oscillations. Maitotoxin activated a nonselective cation current in βTC3 cells that was attenuated by removal of extracellular Na+ and by SKF 96365, in the same manner to a current activated in mouse β-cells following depletion of intracellular Ca2+ stores. Currents similar to these are produced by the mammalian trp-related channels, a gene family that includes Ca2+ store-operated channels and inositol 1,4,5-trisphosphate-activated channels. We found several of the trp family genes were expressed in βTC3 cells by reverse transcriptase polymerase chain reaction using specific primers, but by Northern blot analysis, mtrp-4 was the predominant message expressed. We conclude that a conductance underlying glucose-stimulated oscillations in β-cells is provided by a Ca2+ store depletion-activated nonselective cation current, which is plausibly encoded by homologs of trp genes.

K+ Channels: Generating Excitement in Pancreatic β-Cells

K+ channels play a key role in cellular physiology by regulating the efflux of K+ ions. They are the most diverse group of ion channel proteins; more than 30 K+ channel genes have been characterized. Regulated by ATP, voltage, and calcium, multiple K+ channels coexist in the P-cell to regulate membrane potential, cell excitability, and insulin secretion. Recent developments at the molecular level have greatly expanded our understanding of P-cell K+ channel structure and function, especially in regard to the ATP-sensitive K+ channel, the target for sulfonylurea drugs. Mutations in K+ channel genes underlie diseases as diverse as persistent hyperinsulinemia of infancy, cardiac long QT syndrome, cerebellar degeneration, and certain ataxias. These discoveries have identified new pharmacological targets for possible therapeutic intervention in the treatment of diabetes.

Glucose-induced alterations in β-cell cytoplasmic Ca2+ involving the coupling of intracellular Ca2+ stores and plasma membrane ion channels

The critical event in physiologic glucose-stimulated insulin secretion is the rise, often oscillatory, in intracellular Ca2+ concentration. This has been assumed to be derived exclusively from variations in Ca2+ influx through voltage-dependent Ca2+ channels as a consequence of glucose-induced block of ATP-sensitive K+ channels. Agents that liberate inositol 1,4,5-triphosphate (eg, carbachol) are well known to release Ca2+ from intracellular stores in β cells and islets. Recently, however, evidence has accumulated suggesting an important role for intracellular Ca2+ sequestration and release by the endoplasmic reticulum in the glucose signaling cascade. Moreover, the filling state of the intracellular Ca2+ stores appears to regulate a novel plasma membrane current (Ca2+ release-activated nonselective cation current, /CRAN) whose activity may control glucose-activated membrane potential oscillations and, indirectly, Ca2+ influx and insulin secretion. In this review we consider the evidence supporting these new paradigms for the regulation of intracellular Ca2+ signaling in the β cell and discuss data implicating lesions in these pathways in the pathogenesis of diabetes mellitus.

Single Particle Image Reconstruction of the Human Recombinant Kv2.1 Channel

Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K+ currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic beta-cells. They are therefore an important drug target for the treatment of Type II diabetes mellitus. We used electron microscopy and single particle image analysis to derive a three-dimensional density map of recombinant human Kv2.1. The tetrameric channel is egg-shaped with a diameter of approximately 80 A and a long axis of approximately 120 A. Comparison to known crystal structures of homologous domains allowed us to infer the location of the cytoplasmic and transmembrane assemblies. There is a very good fit of the Kv1.2 crystal structure to the assigned transmembrane assembly of Kv2.1. In other low-resolution maps of K+ channels, the cytoplasmic N-terminal and transmembrane domains form separate rings of density. In contrast, Kv2.1 displays contiguous density that connects the rings, such that there are no large windows between the channel interior and the cytoplasmic space. The crystal structure of KcsA is thought to be in a closed conformation, and the good fit of the KcsA crystal structure to the Kv2.1 map suggests that our preparations of Kv2.1 may also represent a closed conformation. Substantial cytoplasmic density is closely associated with the T1 tetramerization domain and is ascribed to the approximately 184 kDa C-terminal regulatory domains within each tetramer.