Stichodactyla helianthusPeptide, a Pharmacological Tool for Studying Kv3.2 Channels

Abstract

Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well-characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. Chinese hamster ovary (CHO)-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. Stichodactyla helianthus peptide (ShK), isolated from S. helianthus venom and a known high-affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit 86 Rb ϩ efflux from CHO-K1.hKv3.2b (IC 50 ϳ 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus laevis oocytes or in planar patch-clamp studies, ShK inhibited hKv3.2b channels with IC 50 values of ϳ0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic ␤ cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with the blockade of Kv3 channels (i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential after hyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections). Taken together, these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations.

Pharmacological Profiling of Orthochirus scrobiculosus Toxin 1 Analogs with a Trimmed N-Terminal Domain

“…In addition, AOSK1 displays an even larger pharmacological profile through its additional action on Kv3.2. Therefore, AOSK1 is, to our knowledge, the second Kv3.2 channel blocker reported hitherto, after the sea anemone toxin ShK acting in the low nanomolar concentration range (Yan et al, 2005). Here, a strategy of domain trimming was used for the first time to generate new pharmacological profiles.…”

Ion channels in T lymphocytes: An update on facts, mechanisms and therapeutic targeting in autoimmune diseases

“…The evolution of a native peptide into a potent, highly selective, chemically stable, non-toxic and nonimmunogenic molecule is best exemplified by the sea anemone toxin ShK from Stichodactyla helianthus. The native peptide potently blocks Kv1.3 channels along with Kv1.1, Kv1.4, Kv1.6 and Kv3.2 channels and in an effort to improve its selectivity the lysine residue that is highly conserved among K + channel blocking toxins was replaced by the non-natural amino acid diaminopropionic acid [35][36][37]. Although the mutant toxin (ShK-Dap22) displayed improved selectivity for Kv1.3, its efficacy to inhibit T cell activation was later shown to be much lower than that of ShK, and it also turned out to block heterotetramers of Kv1.1 and 1.2 subunits [38].…”

Blockers of the Delayed-Rectifier Potassium Current in Pancreatic β-Cells Enhance Glucose-Dependent Insulin Secretion

“…IonWorks 384-well automated electrophysiology. K V currents were recorded using the IonWorks HT system (Molecular Devices, Sunnyvale, CA) as described previously (21,24). Currents were measured before and 10 min after the addition of GxTX-1E in D-PBS containing 0.03% BSA.…”

“…presence of test compound was added, and incubation proceeded for additional 10 min. 86 Rb ϩ efflux was quantitated as previously described (21). Patch-clamp electrophysiology.…”

“…Xenopus oocyte electrophysiology. Xenopus laevis oocytes were prepared and injected with RNA encoding hK V 2.1, and currents were recorded using two-electrode voltage clamp as described previously (21,23). IonWorks 384-well automated electrophysiology.…”