Robert J. Mertz

Cloning, expression, and distribution of functionally distinct Ca2+-activated K+ channel isoforms from human brain

We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

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.

Insulin‐secreting β‐cells possess specific receptors for interleukin‐1β

The effect of the cytokine interleukin‐1β on the insulin secretory responsiveness of single β‐cells (HIT‐T15) was investigated. In the short‐term, IL‐1β induced a dosage‐dependent stimulation of insulin release. In contrast, in the long‐term, IL‐1β, inhibited both basal and secretagogue‐stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL‐1β on β‐cells. IL‐1β, which has been implicated in the pathogenesis of insulin‐dependent diabetes, may therefore mediate its opposing effects on β‐cells through a specific plasma membrane receptor.

Interleukin-1β inhibits glucokinase activity in clonal HIT-T15 β-cells

Interleukin‐1β (IL‐1β) has been implicated in the pathogenesis of insulin‐dependent diabetes mellitus. In the present study we have investigated the effects of IL‐1β on glucose metabolism in clonal HIT‐T15 β cells. In the short‐term (1 h), 25 IL‐1β significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL‐1β inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6‐phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL‐1β may be due to impaired glucokinase activity.

Synthesis and evaluation of 7-substituted-3-cyclobutylamino-4H-1,2,4-benzothiadiazine-1,1-dioxide derivatives as KATP channel agonists

A series of 7-substituted-3-cyclobutylamino-4H-1,2,4-benzothiadiazine-1,1-dioxide derivatives has been synthesized and evaluated as K(ATP) channel agonists using the inside-out excised patch clamp technique. The most active compounds were approximately 20-fold more potent than diazoxide in opening K(ATP) channels. A linear relationship exists between the potency of the compound and the sigma value of the 7-substituent with electron-withdrawing groups exhibiting higher activity. These compounds may be useful in modulating insulin release from pancreatic beta-cells and in diseases associated with hyperinsulinemia.