Guangxitoxin-1E

The voltage-gated potassium channels Kv2.1 & Kv2.2 are highly expressed in pancreatic islets, yet their contribution to islet hormone secretion is not fully understood. Here we investigate the role of Kv2 channels in pancreatic islets using a combination of genetic & pharmacologic approaches. Pancreatic β-cells from Kv2.1(-/-) mice possess reduced Kv current & display greater glucose-stimulated insulin secretion (GSIS) relative to WT β-cells. Inhibition of Kv2.x channels with selective peptidyl [guangxitoxin-1E (GxTX-1E)] or small molecule (RY796) inhibitors enhances GSIS in isolated wild-type (WT) mouse & human islets, but not in islets from Kv2.1(-/-) mice. However, in WT mice neither inhibitor improved glucose tolerance in vivo. GxTX-1E & RY796 enhanced somatostatin release in isolated human & mouse islets & in situ perfused pancreata from WT & Kv2.1(-/-) mice. Kv2.2 silencing in mouse islets by adenovirus-small hairpin RNA (shRNA) specifically enhanced islet somatostatin, but not insulin, secretion. In mice lacking somatostatin receptor 5, GxTX-1E stimulated insulin secretion & improved glucose tolerance. Collectively, these data show that Kv2.1 regulates insulin secretion in β-cells & Kv2.2 modulates somatostatin release in δ-cells. Development of selective Kv2.1 inhibitors without cross inhibition of Kv2.2 may provide new avenues to promote GSIS for the treatment of type 2 diabetes.

Regulation of delayed rectifier-type K(+) channels (Kv-channels) by glucose was studied in rat pancreatic beta-cells. The Kv-channel current was increased in amplitudes by increasing glucose concentration from 2.8 to 16.6mM, while it was decreased by 2.8mM glucose in a reversible manner (down-regulation) in both perforated & conventional whole-cell modes. The current was decreased by FCCP, intrapipette 0mM ATP or AMPPNP. Glyceraldehyde, pyruvic acid, 2-ketoisocaproic acid, & 10mM MgATP prevented the down-regulation induced by 2.8mM or less glucose. The residual current after treatment with Kv2.1-specific blocker, guangxitoxin-1E, was unchanged by lowering or increasing glucose concentration. We conclude that glucose metabolism regulates Kv2.1 channels in rats beta-cells via altering MgATP levels.

The presence of serum in biological samples often negatively impacts the quality of in vitro assays. However, assays tolerant of serum are useful for assessing the in vivo availability of a small molecule for its target. Electrophysiology assays of ion channels are notoriously sensitive to serum because of their reliance on the interaction of the plasma membrane with a recording electrode. Here we investigate the tolerance of an automated electrophysiology assay for a voltage-gated potassium (K(V)) channel to serum & purified plasma proteins. The delayed rectifier channel, K(V)2.1, stably expressed in Chinese hamster ovary cells produces large, stable currents on the IonWorks Quattro platform (MDS Analytical Technologies, Sunnyvale, CA), making it an ideal test case. K(V)2.1 currents recorded on this platform are highly resistant to serum, allowing recordings in as high as 33% serum. Using a set of compounds related to the K(V) channel blocker, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone, we show that shifts in compound potency with whole serum or isolated serum proteins can be reliably measured with this assay. Importantly, this assay is also relatively insensitive to plasma, allowing the creation of a bioassay for inhibitors of K(V)2.1 channel activity. Here we show that such a bioassay can quantify the levels of the gating modifier, guangxitoxin-1E, in plasma samples from mice dosed with the peptide. This study demonstrates the utility of using an automated electrophysiology platform for measuring serum shifts & for bioassays of ion channel modulators.

Pancreatic beta-cells depolarize in response to glucose & fire calcium-dependent actions potentials that trigger insulin secretion. The major current responsible for action potential repolarization in these cells is a delayed rectifier & Kv2.1 subunits are thought be a major contributor of the delayed rectifier channels. Hence, blockers of Kv2.1 channels might prolong action potentials & enhance calcium influx & insulin secretion. However, the lack of specific small molecule Kv2.1 inhibitors has hindered the testing of this mechanism. Importantly, several gating modifier peptides inhibit Kv2.1 channels in a relatively specific fashion. Hanatoxin (HaTX) & guangxitoxin-1 (GxTX-1) are examples that have been used to probe the role of Kv2.1 channels in beta-cell physiology. Both HaTX & GxTX-1 strongly inhibit the Kv current of beta-cells from various species, arguing that Kv2.1 subunits contribute significantly to the beta-cell delayed rectifier. GxTX-1 prolongs glucose-triggered action potentials, enhances glucose-dependent intracellular calcium elevations & augments glucose-dependent insulin secretion. Taken together, these data suggest that blockers of Kv2.1 channels may be a useful approach to the design of novel therapeutic agents for the treatment of type 2 diabetes. These studies highlight the utility of gating modifier peptides in the study of physiological systems.

Voltage-gated outward K(+) currents from pancreatic islet beta-cells are known to repolarize the action potential during a glucose stimulus, & consequently to modulate Ca(2+) entry & insulin secretion. The voltage gated K(+) (Kv) channel, Kv2.1, which is expressed in rat islet beta-cells, mediates over 60% of the Kv outward K(+) currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25(1-180) (S180), an N-terminal SNAP-25 domain, but not SNAP-25(1-206) (S206), inhibits Kv current & enhances glucose-dependent insulin secretion from rat pancreatic islet beta-cells, & furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.

Delayed-rectifier K+ currents (I(DR)) in pancreatic beta-cells are thought to contribute to action potential repolarization & thereby modulate insulin secretion. The voltage-gated K+ channel, K(V)2.1, is expressed in beta-cells, & the biophysical characteristics of heterologously expressed channels are similar to those of I(DR) in rodent beta-cells. A novel peptidyl inhibitor of K(V)2.1/K(V)2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration approximately 1 nmol/l), has been purified, characterized, & used to probe the contribution of these channels to beta-cell physiology. In mouse beta-cells, GxTX-1 inhibits 90% of I(DR) &, as for K(V)2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the beta-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the beta-cell I(DR), which may provide advantages over currently used therapies for the treatment of type 2 diabetes.