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The sigma-1 receptor (σ-1R) is an endoplasmic reticulum resident chaperone protein involved in a plethora of cellular functions, and whose disruption has been implicated in a wide range of diseases. Genetic analysis has revealed two σ-1R mutants involved in neuromuscular disorders. A point mutation (E102Q) in the ligand-binding domain results in the juvenile form of amyotrophic lateral sclerosis (ALS16), and a 20 amino-acid deletion (Δ31-50) in the putative cytosolic domain leads to a form of distal hereditary motor neuropathy. We investigated the localization and functional properties of these mutants in cell lines using confocal imaging and electrophysiology. The σ-1R mutants exhibited a significant increase in mobility, aberrant localization, and enhanced block of the inwardly rectifying K(+) channel Kir2.1, compared with the wild-type σ-1R. Thus, these σ-1R mutants have different functional properties that could contribute to their disease phenotypes.

Heteropoda venatoria toxin 2 (HpTx2) is an inhibitor cystine knot (ICK)-gating modifier toxin that selectively inhibits Kv4 channels. To characterize the molecular determinants of interaction, we performed alanine scanning of the Kv4.3 S3b region. HpTx2-Kv4.3 interaction had an apparent K(d) value of 2.3 microM. Two alanine mutants in Kv4.3 increased K(d) values to 6.4 microM for V276A and 25 microM for L275A. Simultaneous mutation of both amino acids to alanine nearly eliminated toxin interaction. Unlike Hanatoxin and other well characterized ICK toxins, HpTx2 binding does not require a charged amino acid for interaction. To determine whether the identity of the S3b binding site amino acids altered HpTx2 specificity, we constructed Kv4.3 [LV275IF]. This mutation decreased the K(d) value to 0.54 microM, suggesting that the hydrophobic character of the putative binding site is the most important property for interaction with HpTx2. One mutant, N280A, caused stronger interaction of HpTx2 with Kv4.3; the K(d) value for Kv4.3 [N280A] was 0.26 microM. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 coexpressed with KChIP2b. The toxin's K(d) value for Kv4.3 + KChIP2b was 0.95 microM. KChIP2b stabilizes the closed state of Kv4.3, suggesting that the increased toxin affinity is due to increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins but that the interventions that increase toxin affinity suggest flexibility toward channel binding that belies its unusual specificity for Kv4 channels.

We have hypothesized that the plasma membrane protein components of the exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex, syntaxin 1A and SNAP-25, distinctly regulate different voltage-gated K+ (Kv) channels that are differentially distributed. Neuroendocrine islet cells (alpha, beta, delta) uniformly contain both syntaxin 1A and SNAP-25. However, using immunohistochemistry, we show that the different pancreatic islet cells contain distinct dominant Kv channels, including Kv2.1 in beta cells and Kv2.2 in alpha and delta cells, whose interactions with the SNARE proteins would, respectively regulate insulin, glucagon and somatostatin secretion. We therefore examined the regulation by syntaxin 1A and SNAP-25 of these two channels. We have shown that Kv2.1 interacts with syntaxin 1A and SNAP-25 and, based on studies in oocytes, suggested a model of two distinct modes of interaction of syntaxin 1A and the complex syntaxin 1A/SNAP-25 with the C terminus of the channel. Here, we characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.2 which is highly homologous to Kv2.1, except for the C-terminus. Comparative two-electrode voltage clamp analysis in oocytes between Kv2.2 and Kv2.1 shows that Kv2.2 interacts only with syntaxin 1A and, in contrast to Kv2.1, it does not interact with the syntaxin 1A/SNAP-25 complex and hence is not sensitive to the assembly/disassembly state of the complex. The distinct regulation of these closely related channels by SNAREs may be attributed to differences in their C termini. Together with the differential distribution of these channels among islet cells, their distinct regulation suggests that the documented profound down-regulation of islet SNARE levels in diabetes could distort islet cell ion channels and secretory responses in different ways, ultimately contributing to the abnormal glucose homeostasis.

Kv2.1, the prevalent delayed-rectifier K(+) channel in neuroendocrine and endocrine cells, was suggested previously by our group to be modulated in islet beta-cells by syntaxin 1A (Syx) and soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25). We also demonstrated physical interactions in neuroendocrine cells between Kv2.1, Syx, and SNAP-25, characterized their effects on Kv2.1 activation and inactivation in Xenopus laevis oocytes, and suggested that they pertain to the assembly/disassembly of the Syx/SNAP-25 (t-SNARE) complex. In the present work, we established the existence of a causal relationship between the physical and the functional interactions of Syx with the Kv2.1 channel using three different peptides that compete with the channel for binding of Syx when injected into oocytes already coexpressing Syx with Kv2.1 in the plasma membrane: one peptide corresponding to the Syx-binding region on the N-type Ca(2+) channel, and two peptides corresponding to Syx-binding regions on the Kv2.1 C terminus. All peptides reversed the effects of Syx on Kv2.1, suggesting that the hyperpolarizing shifts of the steady-state inactivation and activation of Kv2.1 caused by Syx result from cell-surface protein-protein interactions and point to participation of the C terminus in such an interaction. In line with these findings, the effects of Syx were dissipated by partial deletions of the C terminus. Furthermore, the t-SNARE complex was shown to bind to the Kv2.1 C terminus, and its effects on the inactivation of Kv2.1 were dissipated by partial deletions of the C terminus. Taken together, these findings suggest that physical interactions of both Syx and the t-SNARE complex with the C terminus of Kv2.1 are involved in channel regulation.

Three novel peptides with the ability to inhibit voltage-dependent potassium channels in the shab (Kv2) and shal (Kv4) subfamilies were identified from the venom of the African tarantulas Stromatopelma calceata (ScTx1) and Heteroscodra maculata (HmTx1, HmTx2). The three toxins are 34- to 38-amino acid peptides that belong to the structural family of inhibitor cystine knot spider peptides reticulated by three disulfide bridges. Electrophysiological recordings in COS cells show that these toxins act as gating modifier of voltage-dependent K+ channels. ScTx1 is the first high-affinity inhibitor of the Kv2.2 channel subtype (IC50, 21.4 nM) to be described. ScTx1 also inhibits the Kv2.1 channels, with an IC50 of 12.7 nM, and Kv2.1/Kv9.3 heteromultimers that have been proposed to be involved in O2 sensing in pulmonary artery myocytes. In addition, it is the most effective inhibitor of Kv4.2 channels described thus far, with an IC50 of 1.2 nM. HmTx toxins share sequence similarities with both the potassium channel blocker toxins (HmTx1) and the calcium channel blocker toxin omega-GsTx SIA (HmTx2). They inhibit potassium current associated with Kv2 subtypes in the 100 to 300 nM concentration range. HmTx2 seems to be a specific inhibitor of Kv2 channels, whereas HmTx1 also inhibits Kv4 channels, including Kv4.1, with the same potency. HmTx1 is the first described peptide effector of the Kv4.1 subtype. Those novel toxins are new tools for the investigation of the physiological role of the different potassium channel subunits in cellular physiology.

Identification of selective ion channel inhibitors represents a critical step for understanding the physiological role that these proteins play in native systems. In particular, voltage-gated potassium (K(V)2) channels are widely expressed in tissues such as central nervous system, pancreas, and smooth muscle, but their particular contributions to cell function are not well understood. Although potent and selective peptide inhibitors of K(V)2 channels have been characterized, selective small molecule K(V)2 inhibitors have not been reported. For this purpose, high-throughput automated electrophysiology (IonWorks Quattro; Molecular Devices, Sunnyvale, CA) was used to screen a 200,000-compound mixture (10 compounds per sample) library for inhibitors of K(V)2.1 channels. After deconvolution of 190 active samples, two compounds (A1 and B1) were identified that potently inhibit K(V)2.1 and the other member of the K(V)2 family, K(V)2.2 (IC(50), 0.1-0.2 μM), and that possess good selectivity over K(V)1.2 (IC(50) >10 μM). Modeling studies suggest that these compounds possess a similar three-dimensional conformation. Compounds A1 and B1 are >10-fold selective over Na(V) channels and other K(V) channels and display weak activity (5-9 μM) on Ca(V) channels. The biological activity of compound A1 on native K(V)2 channels was confirmed in electrophysiological recordings of rat insulinoma cells, which are known to express K(V)2 channels. Medicinal chemistry efforts revealed a defined structure-activity relationship and led to the identification of two compounds (RY785 and RY796) without significant Ca(V) channel activity. Taken together, these newly identified channel inhibitors represent important tools for the study of K(V)2 channels in biological systems.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.

The effects of the antiarrhythmic drug propafenone at Kv2.1 channels were studied with wild-type and mutated channels expressed in Xenopus laevis oocytes. Propafenone decreased the Kv2.1 currents in a time- and voltage-dependent manner (decrease of the time constants of current rise, increase of block with the duration of voltage steps starting from a block of less than 19%, increase of block with the amplitude of depolarization yielding a fractional electrical distance delta of 0.11 to 0.16). Block of Kv2.1 appeared with application to the intracellular, but not the extracellular, side of membrane patches. In mutagenesis experiments, all parts of the Kv2.1 channel were successively exchanged with those of the Kv1.2 channel, which is much more sensitive to propafenone. The intracellular amino and carboxyl terminus and the intracellular linker S4-S5 reduced the blocking effect of propafenone, whereas the linker S5-S6, as well as the segment S6 of the Kv1.2 channel, abolished it to the value of the Kv1.2 channel. In the linker S5-S6, this effect could be narrowed down to two groups of amino acids (groups 372 to 374 and 383 to 384), which also affected the sensitivity to tetraethylammonium. In segment S6, several amino acids in the intracellularly directed part of the helix significantly reduced propafenone sensitivity. The results suggest that propafenone blocks the Kv2.1 channel in the open state from the intracellular side by entering the inner vestibule of the channel. These results are consistent with a direct interaction of propafenone with the lower part of the pore helix and/or residues of segment S6.

The voltage-gated delayed-rectifier-type K+ channel Kv2.1 is expressed in high-density clusters on the soma and proximal dendrites of mammalian central neurons; thus, dynamic regulation of Kv2.1 would be predicted to have an impact on dendritic excitability. Rat brain Kv2.1 polypeptides are phosphorylated extensively, leading to a dramatically increased molecular mass on sodium dodecyl sulfate gels. Phosphoamino acid analysis of Kv2.1 expressed in transfected cells and labeled in vivo with 32P shows that phosphorylation was restricted to serine residues and that a truncation mutant, DeltaC318, which lacks the last 318 amino acids in the cytoplasmic carboxyl terminus, was phosphorylated to a much lesser degree than was wild-type Kv2.1. Whole-cell patch-clamp studies showed that the voltage-dependence of activation of DeltaC318 was shifted to more negative membrane potentials than Kv2.1 without differences in macroscopic kinetics; however, the differences in the voltage-dependence of activation between Kv2.1 and DeltaC318 were eliminated by in vivo intracellular application of alkaline phosphatase, suggesting that these differences were due to differential phosphorylation. Similar analyses of other truncation and point mutants indicated that the phosphorylation sites responsible for the observed differences in voltage-dependent activation lie between amino acids 667 and 853 near the distal end of the Kv2.1 carboxyl terminus. Together, these parallel biochemical and electrophysiological results provide direct evidence that the voltage-dependent activation of the delayed-rectifier K+ channel Kv2. 1 can be modulated by direct phosphorylation of the channel protein; such modulation of Kv2.1 could dynamically regulate dendritic excitability.