Different sensitivity of action potential generation to the rate of depolarization in vagal afferent A-fiber versus C-fiber neurons.

This study demonstrates that the action potential discharge in vagal afferent A-fiber neurons is about 20 times more sensitive to the rate of membrane depolarization compared to C-fiber neurons. The sensitivity of action potential generation to the depolarization rate in vagal sensory neurons is independent of the intensity of current stimuli but nearly abrogated by inhibiting the D-type potassium channel. These findings help better understand the mechanisms that control the activation of vagal afferent nerves.

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

Pore-modulating toxins exploit inherent slow inactivation to block K+ channels.

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

In Silico Analysis of the Subtype Selective Blockage of KCNA Ion Channels through the µ-Conotoxins PIIIA, SIIIA, and GIIIA.

Understanding subtype specific ion channel pore blockage by natural peptide-based toxins is crucial for developing such compounds into promising drug candidates. Herein, docking and molecular dynamics simulations were employed in order to understand the dynamics and binding states of the µ-conotoxins, PIIIA, SIIIA, and GIIIA, at the voltage-gated potassium channels of the KV1 family, and they were correlated with their experimental activities recently reported by Leipold et al. Their different activities can only adequately be understood when dynamic information about the toxin-channel systems is available. For all of the channel-bound toxins investigated herein, a certain conformational flexibility was observed during the molecular dynamic simulations, which corresponds to their bioactivity. Our data suggest a similar binding mode of µ-PIIIA at KV1.6 and KV1.1, in which a plethora of hydrogen bonds are formed by the Arg and Lys residues within the α-helical core region of µ-PIIIA, with the central pore residues of the channel. Furthermore, the contribution of the K+ channel's outer and inner pore loops with respect to the toxin binding. and how the subtype specificity is induced, were proposed.

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels.

We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.

SK Channels Regulate Resting Properties and Signaling Reliability of a Developing Fast-Spiking Neuron.

Reliable and precise signal transmission is essential in circuits of the auditory brainstem to encode timing with submillisecond accuracy. Globular bushy cells reliably and faithfully transfer spike signals to the principal neurons of the medial nucleus of the trapezoid body (MNTB) through the giant glutamatergic synapse, the calyx of Held. Thus, the MNTB works as a relay nucleus that preserves the temporal pattern of firing at high frequency. Using whole-cell patch-clamp recordings, we observed a K+ conductance mediated by small-conductance calcium-activated potassium (SK) channels in the MNTB neurons from rats of either sex. SK channels were activated by intracellular Ca2+ sparks and mediated spontaneous transient outward currents in developing MNTB neurons. SK channels were also activated by Ca2+ influx through voltage-gated Ca2+ channels and synaptically activated NMDA receptors. Blocking SK channels with apamin depolarized the resting membrane potential, reduced resting conductance, and affected the responsiveness of MNTB neurons to signal inputs. Moreover, SK channels were activated by action potentials and affected the spike afterhyperpolarization. Blocking SK channels disrupted the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons and induced extra postsynaptic action potentials in response to presynaptic firing. These data reveal that SK channels play crucial roles in regulating the resting properties and maintaining reliable signal transmission of MNTB neurons.SIGNIFICANCE STATEMENT Reliable and precise signal transmission is required in auditory brainstem circuits to localize the sound source. The calyx of Held synapse in the mammalian medial nucleus of the trapezoid body (MNTB) plays an important role in sound localization. We investigated the potassium channels that shape the reliability of signal transfer across the calyceal synapse and observed a potassium conductance mediated by small-conductance calcium-activated potassium (SK) channels in rat MNTB principal neurons. We found that SK channels are tonically activated and contribute to the resting membrane properties of MNTB neurons. Interestingly, SK channels are transiently activated by calcium sparks and calcium influx during action potentials and control the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons.

Conorfamide-Sr3, a structurally novel specific inhibitor of the Shaker K+ channel.

Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.

The role of axonal Kv1 channels in CA3 pyramidal cell excitability.

Axonal ion channels control spike initiation and propagation along the axon and determine action potential waveform. We show here that functional suppression of axonal Kv1 channels with local puff of dendrotoxin (DTx), laser or mechanical axotomy significantly increased excitability measured in the cell body. Importantly, the functional effect of DTx puffing or axotomy was not limited to the axon initial segment but was also seen on axon collaterals. In contrast, no effects were observed when DTx was puffed on single apical dendrites or after single dendrotomy. A simple model with Kv1 located in the axon reproduced the experimental observations and showed that the distance at which the effects of axon collateral cuts are seen depends on the axon space constant. In conclusion, Kv1 channels located in the axon proper greatly participate in intrinsic excitability of CA3 pyramidal neurons. This finding stresses the importance of the axonal compartment in the regulation of intrinsic neuronal excitability.

Subtype-specific block of voltage-gated K+ channels by µ-conopeptides.

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel.

Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K+-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

Gambierol and n-alkanols inhibit Shaker Kv channel via distinct binding sites outside the K(+) pore.

The marine polycyclic-ether toxin gambierol and 1-butanol (n-alkanol) inhibit Shaker-type Kv channels by interfering with the gating machinery. Competition experiments indicated that both compounds do not share an overlapping binding site but gambierol is able to affect 1-butanol affinity for Shaker through an allosteric effect. Furthermore, the Shaker-P475A mutant, which inverses 1-butanol effect, is inhibited by gambierol with nM affinity. Thus, gambierol and 1-butanol inhibit Shaker-type Kv channels via distinct parts of the gating machinery.

The antifungal plant defensin AtPDF2.3 from Arabidopsis thaliana blocks potassium channels.

Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαß-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked.

KV1 and KV3 Potassium Channels Identified at Presynaptic Terminals of the Corticostriatal Synapses in Rat.

In the last years it has been increasingly clear that KV-channel activity modulates neurotransmitter release. The subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release. To investigate the role of KV in corticostriatal synapses modulation, we combined extracellular recording of population-spike and pharmacological blockage with specific and nonspecific blockers to identify several families of KV channels. We induced paired-pulse facilitation (PPF) and studied the changes in paired-pulse ratio (PPR) before and after the addition of specific KV blockers to determine whether particular KV subtypes were located pre- or postsynaptically. Initially, the presence of KV channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine; in both cases we observed a decrease in PPR that was dose dependent. Further experiments with tityustoxin, margatoxin, hongotoxin, agitoxin, dendrotoxin, and BDS-I toxins all rendered a reduction in PPR. In contrast heteropodatoxin and phrixotoxin had no effect. Our results reveal that corticostriatal presynaptic KV channels have a complex stoichiometry, including heterologous combinations KV1.1, KV1.2, KV1.3, and KV1.6 isoforms, as well as KV3.4, but not KV4 channels. The variety of KV channels offers a wide spectrum of possibilities to regulate neurotransmitter release, providing fine-tuning mechanisms to modulate synaptic strength.

Α-Dendrotoxin-sensitive Kv1 channels contribute to conduction failure of polymodal nociceptive C-fibers from rat coccygeal nerve.

It is known that some patients with diabetic neuropathy are usually accompanied by abnormal painful sensations. Evidence has accumulated that diabetic neuropathic pain is associated with the hyperexcitability of peripheral nociceptors. Previously, we demonstrated that reduced conduction failure of polymodal nociceptive C-fibers and enhanced voltage-dependent sodium currents of small dorsal root ganglion (DRG) neurons contribute to diabetic hyperalgesia. To further investigate whether and how potassium channels are involved in the conduction failure, α-dendrotoxin (α-DTX), a selective blocker of the low-threshold sustained Kv1 channel, was chosen to examine its functional capability in modulating the conduction properties of polymodal nociceptive C-fibers and the excitability of sensory neurons. We found that α-DTX reduced the conduction failure of C-fibers from coccygeal nerve in vivo accompanied by an increased initial conduction velocity but a decreased activity-dependent slowing of conduction velocity. In addition, the number of APs evoked by step currents was significantly enhanced after the treatment with α-DTX in small-diameter sensory neurons. Further study of the mechanism indicates α-DTX-sensitive K(+) current significantly reduced and the activation of this current in peak and steady state shifted to depolarization for diabetic neurons. Expression of Kv channel subunits Kv1.2 and Kv1.6 was downregulated in both small dorsal root ganglion neurons and peripheral C-fibers. Taken together, these results suggest that α-DTX-sensitive Kv1 channels might play an important role in regulating the conduction properties of polymodal nociceptive C-fibers and firing properties of sensory neurons.

Alkanols inhibit voltage-gated K(+) channels via a distinct gating modifying mechanism that prevents gate opening.

Alkanols are small aliphatic compounds that inhibit voltage-gated K(+) (K(v)) channels through a yet unresolved gating mechanism. K(v) channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker K(v) channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type K(v) channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state.

Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons.

Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder.

New inhibitors of the Kvß2 subunit from mammalian Kv1 potassium channels.

The role of the redox state of Kvß subunits in the modulation of Kv1 potassium channels has been well documented over the past few years. It has been suggested that a molecule that binds to or inhibits the aldo-keto reductase activity of Kvß might affect the modulation of channel properties. Previous studies of possible modulators of channel activity have shown that cortisone and some related compounds are able to physically dissociate the channel components by binding to a site at the interface between α and ß subunits. Herein, we describe some new inhibitors of rat brain Kvß2, identified using an assay based on multiple substrate turnover. This approach allows one to focus on molecules that specifically block NADPH oxidation. These studies showed that, at 0.5mM, 3,4-dihydroxphenylacetic acid (DOPAC) was an inhibitor of Kvß2 turnover yielding a ∼ 40-50% reduction in the aldehyde reductase activity of this subunit. Other significant inhibitors include the bioflavinoid, rutin and the polyphenol resveratrol; some of the known cardioprotective effects of these molecules may be attributable to Kv1 channel modulation. Cortisone or catechol caused moderate inhibition of Kvß2 turnover, and the aldo-keto reductases inhibitor valproate had an even smaller effect. Despite the importance of the Kv1 channels in a number of disease states, there have been few Kvß2 inhibitors reported. While the ones identified in this study are only effective at high concentrations, they could serve as tools to decipher the role of Kvß2 in vivo and, eventually, inform the development of novel therapeutics.

Mutations in the S6 gate isolate a late step in the activation pathway and reduce 4-AP sensitivity in shaker K(v) channel.

Kv channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10-20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker's sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.