Jennings F. Worley

IonWorks™ HT: A New High-Throughput Electrophysiology Measurement Platform

To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a plate-based electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate™). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate™, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks™ HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening. (Journal of Biomolecular Screening 2003:50-64)

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.

HTS approaches to voltage-gated ion channel drug discovery

Voltage-gated ion channels are emerging as a target class of increasing importance to the pharmaceutical industry because of their relevance to a wide variety of diseases in the cardiovascular, CNS and metabolic areas. In the quest to identify novel lead molecules against these targets, drug discovery programmes are increasingly making use of HTS approaches. The authors describe the current technologies available for voltage-gated ion-channel screening, their application to HTS campaigns and the current limitations and emerging technologies within this area.

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.

Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction.

A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.

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.

An industrial perspective on utilizing functional ion channel assays for high throughput screening.

The ability to apply large-scale screening formats to measures of ion channel function offers immense opportunities for drug discovery and academic research. Technologies have been developed over the last several years that now provide the ability to screen large numbers of compounds and natural products on ion channel function to find novel drugs. Application of these technologies has vastly improved the capabilities of ion channel drug discovery and provides an avenue to accelerate discoveries of ion channel biology. These advances have largely arisen from the development and application of instruments and reporters of membrane potential and ion movements in cells used to measure functional activity of ion channels. This article endeavors to describe the practical applications of these technologies in developing, validating, and implementing high throughput screening assay formats to different types of ion channels.

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.