Structure-Activity Analysis of N-Type Calcium Channel Inhibitor SO-3.

As an ω-conopeptide originally discovered from Conus striatus, SO-3 contains 25 amino acid residues and three disulfide bridges. Our previous study has shown that this peptide possesses potent analgesic activity in rodent pain models (mouse and rat), and it specifically inhibits an N-type calcium ion channel (Cav2.2). In the study presented here, we investigated the key amino acid residues for their inhibitory activity against Cav2.2 expressed in HEK 293 cells and analgesic activity in mice. To improve the inhibitory activity of SO-3, we also evaluated the effects of some amino acid residues derived from the corresponding residues of ω-peptide MVIIA, CVID, or GVIA. Our data reveal that Lys6, Ile11, and Asn14 are the important functional amino acid residues for SO-3. The replacement of some amino acid residues of SO-3 in loop 1 with the corresponding residues of CVID and GVIA improved the inhibitory activity of SO-3. The binding mode of Cav2.2 with SO-3 amino acids in loop 1 and loop 2 may be somewhat different from that of MVIIA. This study expanded our knowledge of the structure-activity relationship of ω-peptides and provided a new strategy for improving the potency of Cav2.2 inhibitors.

The residue I257 at S4-S5 linker in KCNQ1 determines KCNQ1/KCNE1 channel sensitivity to 1-alkanols.

AIM: KCNQ1 and KCNE1 form a complex in human ventricular cardiomyocytes, which are important in maintaining a normal heart rhythm. In the present study we investigated the effects of a homologous series of 1-alkanols on KCNQ1/KCNE1 channels expressed in Xenopus oocytes. METHODS: ECG recording was made in rats injected with ethanol-containing solution (0.3 mL, ip). Human KCNQ1 channel and its auxiliary subunit KCNE1 were heterologously coexpressed in Xenopus oocytes, which were superfused with ND96 solution; 1-alkanols (ethanol, 1-butanol and 1-hexanol) were delivered through a gravity-driven perfusion device. The slow-delayed rectifier potassium currents IKs (KCNQ1/KCNE1 currents) were recorded using a two-electrode voltage clamp method. Site-directed mutations (I257A) were made in KCNQ1. RESULTS: In ECG recordings, a low concentration of ethanol (3%, v/v) slightly increased the heart rate of rats, whereas the higher concentrations of ethanol (10%, 50%, v/v) markedly reduced it. In oocytes coexpressing KCNQ1/KCNE1 channels, ethanol, 1-butanol and 1-hexanol dose-dependently inhibited IKs currents with IC50 values of 80, 11 and 2.7 mmol/L, respectively. Furthermore, the 1-alkanols blocked the KCNQ1 channel in both open and closed states, and a four-state model could adequately explain the effects of 1-alkanols on the closed-state channel block. Moreover, the mutation of I257A at the intracellular loop between S4 and S5 in KCNQ1 greatly decreased the sensitivity to 1-alkanols; and the IC50 values of ethanol, 1-butanol and 1-hexanol were increased to 634, 414 and 7.4 mmol/L, respectively. The mutation also caused the ablation of closed-state channel block. CONCLUSION: These findings provide new insight into the intricate mechanisms of the blocking effects of ethanol on the KCNQ1 channel.

Structural basis for calcium and magnesium regulation of a large conductance calcium-activated potassium channel with ß1 subunits.

Large conductance Ca(2+)- and voltage-activated potassium (BK) channels, composed of pore-forming α subunits and auxiliary ß subunits, play important roles in diverse physiological activities. The ß1 is predominately expressed in smooth muscle cells, where it greatly enhances the Ca(2+) sensitivity of BK channels for proper regulation of smooth muscle tone. However, the structural basis underlying dynamic interaction between BK mSlo1 α and ß1 remains elusive. Using macroscopic ionic current recordings in various Ca(2+) and Mg(2+) concentrations, we identified two binding sites on the cytosolic N terminus of ß1, namely the electrostatic enhancing site (mSlo1(K392,R393)-ß1(E13,T14)), increasing the calcium sensitivity of BK channels, and the hydrophobic site (mSlo1(L906,L908)-ß1(L5,V6,M7)), passing the physical force from the Ca(2+) bowl onto the enhancing site and S6 C-linker. Dynamic binding of these sites affects the interaction between the cytosolic domain and voltage-sensing domain, leading to the reduction of Mg(2+) sensitivity. A comprehensive structural model of the BK(mSlo1 α-ß1) complex was reconstructed based on these functional studies, which provides structural and mechanistic insights for understanding BK gating.

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes.

T-superfamily conotoxins have a typical cysteine pattern of "CC-CC", and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4ß2 and α3ß2, with IC50 values of 343.4 and 1047.2nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LßT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4ß2, all demonstrated that the residues Ile(6) and Leu(14) were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4ß2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.

An ATPase inhibitory peptide with antibacterial and ion current effects.

An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35-65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic ß cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.

Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family.

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 µM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani.

Centipedes have venom glands in their first pair of limbs, and their venoms contain a large number of components with different biochemical and pharmacological properties. However, information about the compositions and functions of their venoms is largely unknown. In this study, Scolopendra subspinipes dehaani venoms were systematically investigated by transcriptomic and proteomic analysis coupled with biological function assays. After random screening approximately 1500 independent clones, 1122 full length cDNA sequences, which encode 543 different proteins, were cloned from a constructed cDNA library using a pair of venom glands from a single centipede species. Neurotoxins, ion channel acting components and venom allergens were the main fractions of the crude venom as revealed by transcriptomic analysis. Meanwhile, 40 proteins/peptides were purified and characterized from crude venom of S. subspinipes dehaani. The N-terminal amino acid sequencing and mass spectrum results of 29 out of these 40 proteins or peptides matched well with their corresponding cDNAs. The purified proteins/peptides showed different pharmacological properties, including the following: (1) platelet aggregating activity; (2) anticoagulant activity; (3) phospholipase A(2) activity; (4) trypsin inhibiting activity; (5) voltage-gated potassium channel activities; (6) voltage-gated sodium channel activities; (7) voltage-gated calcium channel activities. Most of them showed no significant similarity to other protein sequences deposited in the known public database. This work provides the largest number of protein or peptide candidates with medical-pharmaceutical significance and reveals the toxin nature of centipede S. subspinipes dehaani venom.

Action potential bursts enhance transmitter release at a giant central synapse.

Patterns of action potentials (APs), often in the form of bursts, are critical for coding and processing information in the brain. However, how AP bursts modulate secretion at synapses remains elusive. Here, using the calyx of Held synapse as a model we compared synaptic release evoked by AP patterns with a different number of bursts while the total number of APs and frequency were fixed. The ratio of total release produced by multiple bursts to that by a single burst was defined as 'burst-effect'.We found that four bursts of 25 stimuli at 100 Hz increased the totalcharge of EPSCs to 1.47 ± 0.04 times that by a single burst of 100 stimuli at the same frequency.Blocking AMPA receptor desensitization and saturation did not alter the burst-effect, indicating that it was mainly determined by presynaptic mechanisms. Simultaneous dual recordings of presynaptic membrane capacitance (Cm) and EPSCs revealed a similar burst-effect, being 1.58±0.13by Cm and 1.49±0.05 by EPSCs. Reducing presynapticCa2+ influx by lowering extracellular Ca2+concentration or buffering residual intracellular Ca2+ with EGTA inhibited the burst-effect. We further developed a computational model largely recapitulating the burst-effect and demonstrated that this effect is highly sensitive to dynamic change in availability of the releasable pool of synaptic vesicles during various patterns of activities. Taken together, we conclude that AP bursts modulate synaptic output mainly through intricate interaction between depletion and replenishment of the large releasable pool. This burst-effect differs from the somatic burst-effect previously described from adrenal chromaffin cells, which substantially depends on activity-induced accumulation of Ca2+ to facilitate release of a limited number of vesicles in the releasable pool. Hence, AP bursts may play an important role in dynamically regulating synaptic strength and fidelity during intense neuronal activity at central synapses.

Role of vesicle pools in action potential pattern-dependent dopamine overflow in rat striatum in vivo.

Action potential (AP) patterns and dopamine (DA) release are known to correlate with rewarding behaviors, but how codes of AP bursts translate into DA release in vivo remains elusive. Here, a given AP pattern was defined by four codes, termed total AP number, frequency, number of AP bursts, and interburst time [N, f, b, i].. The 'burst effect' was calculated by the ratio (γ) of DA overflow by multiple bursts to that of a single burst when total AP number was fixed. By stimulating the medial forebrain bundle using AP codes at either physiological (20 Hz) or supraphysiological (80 Hz) frequencies, we found that DA was released from two kinetically distinct vesicle pools, the fast-releasable pool (FRP) and prolonged-releasable pool (PRP), in striatal dopaminergic terminals in vivo. We examined the effects of vesicle pools on AP-pattern dependent DA overflow and found, with given 'burst codes' [b=8, i=0.5 s], a large total AP number [N = 768, f = 80 Hz] produced a facilitating burst-effect (γ[b8/b1] = 126 ± 3%), while a small total AP number [N=96, 80 Hz] triggered a depressing-burst-effect (γ[b8/b1] = 29 ± 4%). Furthermore, we found that the PRP (but not the FRP) predominantly contributed to the facilitating-burst-effect and the FRP played an important role in the depressing-burst effect. Thus, our results suggest that striatal DA release captures pre-synaptic AP pattern information through different releasable pools.

High-density localization of active molecules using Structured Sparse Model and Bayesian Information Criterion.

Localization-based super-resolution microscopy (or called localization microscopy) rely on repeated imaging and localization of active molecules, and the spatial resolution enhancement of localization microscopy is built upon the sacrifice of its temporal resolution. Developing algorithms for high-density localization of active molecules is a promising approach to increase the speed of localization microscopy. Here we present a new algorithm called SSM_BIC for such purpose. The SSM_BIC combines the advantages of the Structured Sparse Model (SSM) and the Bayesian Information Criterion (BIC). Through simulation and experimental studies, we evaluate systematically the performance between the SSM_BIC and the conventional Sparse algorithm in high-density localization of active molecules. We show that the SSM_BIC is superior in processing single molecule images with weak signal embedded in strong background.

Localization-based super-resolution microscopy with an sCMOS camera.

In the community of localization-based super-resolution microscopy (or called localization microscopy), it is generally believed that the emission of single molecules is so weak that an EMCCD (electron multiplying charge coupled device) camera is necessary to be used as the detector by eliminating read noise. Here we evaluate the possibility of a new kind of low light detector, scientific complementary metal-oxide-semiconductor (sCMOS) camera in localization microscopy. We demonstrate experimentally that sCMOS is capable of imaging actin bundles with FWHM diameter of 37 nm, evidencing the capability of sCMOS in localization microscopy. We further characterize the noise performance of sCMOS and find out that, with the use of a bright fluorescence probe such as d2EosFP, localization microscopy imaging is now working in the shot noise limited region.

Fast inactivation of Nav current in rat adrenal chromaffin cells involves two independent inactivation pathways.

Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and tetrodotoxin (TTX)-sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. Here, through recordings of Nav current in rat CCs, primarily in adrenal medullary slices, we describe unique inactivation properties of CC Nav inactivation that help define AP firing rates in CCs. The key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of similar amplitude. Various paired pulse protocols show that entry into the fast and slower recovery processes result from largely independent competing inactivation pathways, each of which occurs with similar onset times at depolarizing potentials. Over voltages from -120 to -80 mV, faster recovery varies from ∼3 to 30 ms, while slower recovery varies from ∼50 to 400 ms. With strong depolarization (above -10 mV), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of short depolarizations favor recovery from fast recovery pathways and result in cumulative increases in the slow recovery fraction. Dual-pathway fast inactivation, by promoting use-dependent accumulation in slow recovery pathways, dynamically regulates Nav availability. Consistent with this finding, repetitive AP clamp waveforms at 1-10 Hz frequencies reduce Nav availability 80-90%, depending on holding potential. These results indicate that there are two distinct pathways of fast inactivation, one leading to conventional fast recovery and the other to slower recovery, which together are well-suited to mediate use-dependent changes in Nav availability.

Author Correction: Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential regulation of action potentials by inactivating and noninactivating BK channels in rat adrenal chromaffin cells.

Large-conductance Ca(2+)-activated K(+) (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca(2+) and membrane potential. In rat chromaffin cells (RCC), inactivating BK(i) and noninactivating (BK(s)) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK(i) and BK(s) current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK(i) current facilitates repetitive firing of APs, whereas BK(s) current does not. However, the key functional difference between BK(i) and BK(s) current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK(i) current at a given [Ca(2+)].

A conserved arginine/lysine-based motif promotes ER export of KCNE1 and KCNE2 to regulate KCNQ1 channel activity.

KCNE ß-subunits play critical roles in modulating cardiac voltage-gated potassium channels. Among them, KCNE1 associates with KCNQ1 channel to confer a slow-activated IKs current, while KCNE2 functions as a dominant negative modulator to suppress the current amplitude of KCNQ1. Any anomaly in these channels will lead to serious myocardial diseases, such as the long QT syndrome (LQTS). Trafficking defects of KCNE1 have been reported to account for the pathogenesis of LQT5. However, the molecular mechanisms underlying KCNE forward trafficking remain elusive. Here, we describe an arginine/lysine-based motif ([R/K](S)[R/K][R/K]) in the proximal C-terminus regulating the endoplasmic reticulum (ER) export of KCNE1 and KCNE2 in HEK293 cells. Notably, this motif is highly conserved in the KCNE family. Our results indicate that the forward trafficking of KCNE2 controlled by the motif (KSKR) is essential for suppressing the cell surface expression and current amplitude of KCNQ1. Unlike KCNE2, the motif (RSKK) in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1. Furthermore, truncations of the C-terminus did not reduce the apparent affinity of KCNE2 for KCNQ1, demonstrating that the rigid C-terminus of KCNE2 may not physically interact with KCNQ1. In contrast, the KCNE1 C-terminus is critical for its interaction with KCNQ1. These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2 membrane targeting and how they coassemble with KCNQ1 to regulate the channels activity.

Rectification ratio based determination of disulfide bonds of ß2 extracellular loop of BK channel.

Large-conductance Ca2+-activated K+ (BK) channels are composed of a pore-forming α and a variable number of auxiliary ß subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The ß2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of ß2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK α to ß2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of ß2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in ß2 subunit in complex with a BK α subunit, which are helpful to predict structural model of ß2 subunits through computational simulation and to understand the interface between the extracellular domain of the ß subunits and the pore-forming α subunit.

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity.

Single large-conductance calcium-activated K(+) (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca(2+) ](in) increased from 10 to 300 microM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a "shutter-like" mechanism gating the permeation of K(+) ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.

Slack and Slick KNa channels are required for the depolarizing afterpotential of acutely isolated, medium diameter rat dorsal root ganglion neurons.

AIM: Na+-activated K+ (K(Na)) channels set and stabilize resting membrane potential in rat small dorsal root ganglion (DRG) neurons. However, whether K(Na) channels play the same role in other size DRG neurons is still elusive. The aim of this study is to identify the existence and potential physiological functions of K(Na) channels in medium diameter (25-35 microm) DRG neurons. METHODS: Inside-out and whole-cell patch-clamp were used to study the electrophysiological characterizations of native K(Na) channels. RT-PCR was used to identify the existence of Slack and Slick genes. RESULTS: We report that K(Na) channels are required for depolarizing afterpotential (DAP) in medium sized rat DRG neurons. In inside-out patches, K(Na) channels represented 201 pS unitary chord conductance and were activated by cytoplasmic Na+ [the half maximal effective concentration (EC50): 35 mmol/L] in 160 mmol/L symmetrical K+o/K+i solution. Additionally, these K(Na) channels also represented cytoplasmic Cl(-)-dependent activation. RT-PCR confirmed the existence of Slack and Slick genes in DRG neurons. Tetrodotoxin (TTX, 100 nmol/L) completely blocked the DRG inward Na+ currents, and the following outward currents which were thought to be K(Na) currents. The DAP was increased when extracellular Na+ was replaced by Li+. CONCLUSION: We conclude that Slack and Slick K(Na) channels are required for DAP of medium diameter rat DRG neurons that regulate DRG action potential repolarization.

Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary ß (ß1-ß4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched ß4 subunit (hß4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hß4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of ß4 subunit's extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by ß4 are still unclear. To address them, here, we first identified four disulfide bonds in hß4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of ß4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/ß4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with ß4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(ß4).

A limited access compartment between the pore domain and cytosolic domain of the BK channel.

Cytosolic N-terminal segments of many K+ channel subunits mediate rapid blockade of ion permeation by physical occlusion of the ion-conducting pore. For some channels with large cytosolic structures, access to the channel pore by inactivation domains may occur through lateral entry pathways or "side portals" that separate the pore domain and associated cytosolic structures covering the axis of the permeation pathway. However, the extent to which side portals control access of molecules to the channel or influence channel gating is unknown. Here we use removal of inactivation by trypsin as a tool to examine basic residue accessibility in both the N terminus of the native auxiliary beta2 subunit of Ca2+-activated, BK-type K+ channels and beta2 subunits with artificial inactivating N termini. The results show that, for BK channels, side portals define a protected space that precedes the channel permeation pathway and excludes small proteins such as trypsin but allows inactivation domains to enter. When channels are closed, inactivation domains readily pass through side portals, with a central antechamber preceding the permeation pathway occupied by an inactivation domain approximately half of the time under resting conditions. The restricted volume of the pathway through side portals is likely to influence kinetic properties of inactivation mechanisms, blockade by large pharmacological probes, and accessibility of modulatory factors to surfaces of the channel within the protected space.