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Hanatoxin (HaTx) from spider venom works as an inhibitor of Kv2.1 channels, most likely by interacting with the voltage sensor (VS). However, the way in which this water-soluble peptide modifies the gating remains poorly understood as the VS is deeply embedded within the bilayer, although it would change its position depending on the membrane potential. To determine whether HaTx can indeed bind to the VS, the depth at which HaTx penetrates into the POPC membranes was measured with neutron reflectivity. Our results successfully demonstrate that HaTx penetrates into the membrane hydrocarbon core (∼9 Å from the membrane surface), not lying on the membrane-water interface as reported for another voltage sensor toxin (VSTx). This difference in penetration depth suggests that the two toxins fix the voltage sensors at different positions with respect to the membrane normal, thereby explaining their different inhibitory effects on the channels. In particular, results from MD simulations constrained by our penetration data clearly demonstrate an appropriate orientation for HaTx to interact with the membranes, which is in line with the biochemical information derived from stopped-flow analysis through delineation of the toxin-VS binding interface.

Membrane perturbation induced by cysteine-rich peptides is a crucial biological phenomenon but scarcely investigated, in particular with effective biophysical-chemical methodologies. Hanatoxin (HaTx), a 35-residue polypeptide from spider venom, works as an inhibitor of drk1 (Kv2.1) channels, most likely by interacting with the voltage-sensor. However, how this water-soluble peptide modifies the gating remains poorly understood, as the voltage sensor was proposed to be deeply embedded within the bilayer. To see how HaTx interacts with phospholipid bilayers, we observe the toxin-induced perturbation on POPC/DOPG-membranes through measurements of the change in membrane thickness. Lamellar X-ray diffraction (LXD) was applied on stacked planar bilayers in the near-fully hydrated state. The results provide quantitative evidence for the membrane thinning in a concentration-dependent manner, leading to novel and direct combinatory approaches by discovering how to investigate such a biologically relevant interaction between gating-modifier toxins and phospholipid bilayers.

The ROMK1 (Kir1.1) channel activity is predominantly regulated by intracellular pH (pHi) and phosphatidylinositol 4,5-bisphosphate (PIP2). Although several residues were reported to be involved in the regulation of pHi associated with PIP2 interaction, the detailed molecular mechanism remains unclear. We perform experiments in ROMK1 pHi-gating with electrophysiology combined with mutational and structural analysis. In the present study, non basic residues of C-terminal region (S219, N215, I192, L216 and L220) in ROMK1 channels have been found to mediate channel-PIP2 interaction and pHi gating. Further, our structural results show these residues with an appropriate distance to interact with membrane PIP2. Meanwhile, a cluster of basic residues (R188, R217 and K218), which was previously discovered regarding the interaction with PIP2, exists in this appropriate distance to discriminate the regulation of channel-PIP2 interaction and pHi-gating. This appropriate distance can be observed with high conservation in the Kir channel family. Our results provide insight that an appropriate distance cooperates with the electrostatics interaction of channel-PIP2 to regulate pHi-gating.

Hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS) is a severe salt-losing renal tubular disorder and results from the mutation of renal outer medullary K(+) (ROMK1) channels. The aberrant ROMK1 function induces alterations in intracellular pH (pH(i)) gating under physiological conditions. We investigate the role of protein kinase A (PKA) in the pH(i) gating of ROMK1 channels. Using giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels, PKA-mediated phosphorylation decreased the sensitivity of ROMK1 channels to pH(i). A homology model of ROMK1 reveals that a PKA phosphorylation site (S219) is spatially juxtaposed to the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding residues (R188, R217, and K218). Molecular dynamics simulations suggest a stable transition state, in which the shortening of distance between S219 and R217 and the movement of K218 towards the membrane after the PKA-phosphorylation can be observed. Such conformational change may bring the PIP(2) binding residues (K218) more accessible to the membrane-bound PIP(2). In addition, PIP(2) dose-dependently reactivates the acidification-induced rundown channels only when ROMK1 channels have been phosphorylated by PKA. This implies a sequence regulatory episode reflecting the role of PIP(2) in the pH(i) gating of ROMK1 channels by PKA-mediated phosphorylation. Our results provide new insights into the molecular mechanisms underlying the ROMK1 channel regulation associated with HPS/aBS.

Background/Purpose: Inward rectifying potassium channel 6.2 (Kir6.2 Δ C26 channel) is closely related to ATP-sensitive potassium channels. Whether sodium azide, barium ion, d-amphetamine or procaine acts directly on the Kir6.2 Δ C26 channel remains unclear. We studied the effects of these compounds on Kir6.2 Δ C26 channel expressed in Xenopus oocytes. Methods: The coding sequence of a truncated form of mouse Kir6.2 (GenBank accession number NP_034732.1), Kir6.21-364 (i.e. Kir6.2 Δ C26), was subcloned into the pET20b(+) vector. Plasmid containing the correct T7 promoter-Kir6.21-364 cDNA fragment [Kir6.2/pET20b(+)] was then subject to Not I digestion to generate the templates for in vitro run-off transcriptions. The channel was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to measure the effects of sodium azide, barium ion, d-amphetamine and procaine on Kir6.2 Δ C26 channel current. Results: Sodium azide activated and barium ion and d-amphetamine inhibited the Kir6.2 Δ C26 channel. Procaine did not have any significant effect on the Kir6.2 Δ C26 channel. Conclusion: Kir6.2 Δ C26 channel expressed in Xenopus oocytes can be used as a pharmacological tool for the study of inward rectifying potassium channels.

The pro-inflammatory cytokines TNF-alpha and IL-1beta are two of the important mediators involved in the several chronic inflammatory diseases. We used the release of TNF-alpha and IL-1beta from lipopolysaccharide-stimulated human PBMC as inflammatory indexes to discover the potential anti-inflammatory candidates. Among near 500 chemical compounds, MT4 had the suppressive action on the release of TNF-alpha and IL-1beta in PBMC with IC50 values of 22 and 44 nM, respectively. After verified the MT4 inhibitory mechanism, the results revealed that p38alpha and p38beta MAPK activity was inhibited by MT4 with an IC50 value of 0.13 and 0.55 microM, respectively. Further characterization of enzyme kinetics showed the binding mode of MT4 was competitive with the ATP substrate-binding site of p38alpha MAPK.

While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175–179). However, if one only relies on such information, more advanced structure–functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3–S4 linker region, we suggested a significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3–S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding-modified gating is proposed. Copyright © 2003 John Wiley & Sons, Ltd.

Rundown is a generally encountered problem while recording KATP channel activity with inside-out patches. No assigned structural fragment related to this mechanism has yet been derived from any of the functional analyses performed. Therefore, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, we propose here the three-dimensional structural model of an intracellular C-terminal domain of the Kir6.2 subunit in KATP channels. An E. coli CMP-kinase was suggested as template for the model building. The subdomain arrangement of this novel kinase domain and the structural correlation for UDP-docking are described. With structural-functional interpretation, we conclude that the reactivation of KATP channel rundown by MgATP or UDP is very possibly regulated by this intracellular kinase domain at the C-terminus of Kir6.2 subunit in KATP channels.

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Delta109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of approximately pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.

The protein kinase C (PKC) pathway is important for the regulation of K+ transport. The renal outer medullar K+ (ROMK1) channels show an exquisite sensitivity to intracellular protons (pH i ) (effective pK a approximately 6.8) and play a key role in K+ homeostasis during metabolic acidosis. Our molecular dynamic simulation results suggest that PKC-mediated phosphorylation on Thr-193 may disrupt the PIP2-channel interaction via a charge–charge interaction between Thr-193 and Arg-188. Therefore, we investigated the role of PKC and pH i in regulation of ROMK1 channel activity using a giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels. ROMK1 channels pre-incubated with the PKC activator phorbol-12-myristate-13-acetate exhibited increased sensitivity to pH i (effective pK a shifted to pH approximately 7.0). In the presence of GF109203X—a PKC selective inhibitor—the effective pK a for inhibition of ROMK1 channels by pH i decreased (effective pK a shifted to pH approximately 6.5). The pH i sensitivity of ROMK1 channels mediated by PKC appeared to be dependent of PIP2 depletion. The giant patch clamp together with site direct mutagenesis revealed that Thr-193 is the phosphorylation site on PKC that regulates the pH i sensitivity of ROMK1 channels. Mutation of PKC-induced phosphorylation sites (T193A) decreases the pH i sensitivity and increases the interaction of channel-PIP2. Taken together, these results provide new insights into the molecular mechanisms underlying the pH i gating of ROMK1 channel regulation by PKC.

Voltage-dependent potassium channel Kv2.1 is widely expressed in mammalian neurons and was suggested responsible for mediating the delayed rectifier (I(K)) currents. Further investigation of the central role of this channel requires the development of specific pharmacology, for instance, the utilization of spider venom toxins. Most of these toxins belong to the same structural family with a short peptide reticulated by disulfide bridges and share a similar mode of action. Hanatoxin 1 (HaTx1) from a Chilean tarantula was one of the earliest discussed tools regarding this and has been intensively applied to characterize the channel blocking not through the pore domain. Recently, more related novel toxins from African tarantulas such as heteroscordratoxins (HmTx) and stromatoxin 1 (ScTx1) were isolated and shown to act as gating modifiers such as HaTx on Kv2.1 channels with electrophysiological recordings. However, further interaction details are unavailable due to the lack of high-resolution structures of voltage-sensing domains in such mammalian Kv channels. Therefore, in the present study, we explored structural observation via molecular docking simulation between toxins and Kv2.1 channels based upon the solution structures of HaTx1 and a theoretical basis of an individual S3(C) helical channel fragment in combination with homology modeling for other novel toxins. Our results provide precise chemical details for the interactions between these tarantula toxins and channel, reasonably correlating the previously reported pharmacological properties to the three-dimensional structural interpretation. In addition, it is suggested that certain subtle structural variations on the interaction surface of toxins may discriminate between the related toxins with different affinities for Kv channels. Evolutionary links between spider peptide toxins and a "voltage sensor paddles" mechanism most recently found in the crystal structure of an archaebacterial K(+) channel, KvAP, are also delineated in this paper.

In the homotrimeric OmpF porin from Escherichia coli , each channel is constricted by a loop protruding into the β-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Δ116–120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.

The sole histidine residue in OmpF porin was replaced by threonine using site-directed mutagenesis. This exchange affected neither channel properties nor channel structure, as determined by X-ray analysis to 3.2 A. Conductance and critical voltage (Vc) were observed in the pH range 4.3-9.4, with results indistinguishable from those observed in the wild-type protein. The validity of these observations is supported by the independence of the methods used, and by the fact that mutants in residues located in the channel constriction yielded significantly different values from wild-type protein. The binding of a glycolipid molecule might be affected.