Putative Binding Sites,and Pathways to Them,for Amidine and Guanidine Current Inhibitors on Acid‐Sensing Ion Channels (ASIC). A Theoretical Approach with hASIC1a Homology Model

Central inhibition of the acid‐sensing hASIC1a channel, acting upstream of the opiate system, might serve to treat any type of pain, avoiding the unwanted addiction problems of the opioid drugs. To this end, inhibition of hASIC1a channel by PcTx1, a peptide from the Trinidad chevron tarantula, is under development. New inhibitors of the hASIC1a channel are also being sought, in the hope of further modulating the activity, from which antiplasmodial amidine and guanidine phenyl drugs have emerged as promising candidates. However, how such current inhibition takes place remains obscure from the molecular point of view, hindering any further progress in developing drugs. Therefore, the nature of the binding sites, and how they are reached by the amidine‐guanidine drugs, was investigated here via automated docking and molecular dynamics with hASIC1a homology models. This study has revealed that this ion channel is rich in binding sites, and that flexible drugs, such as nafamostat, may penetrate it in a snake‐like elongated conformation. Then, crawling like a snake through temporary holes in the protein, nafamostat either simply flips, or changes to a high‐energy folded conformation to become adapted to the shape of the binding site.     

Putative binding sites, and pathways to them, for amidine and guanidine current inhibitors on acid-sensing ion channels (ASIC). A theoretical approach with hASIC1a homology model

Central inhibition of the acid-sensing hASIC1a channel, acting upstream of the opiate system, might serve to treat any type of pain, avoiding the unwanted addiction problems of the opioid drugs. To this end, inhibition of hASIC1a channel by PcTx1, a peptide from the Trinidad chevron tarantula, is under development. New inhibitors of the hASIC1a channel are also being sought, in the hope of further modulating the activity, from which antiplasmodial amidine and guanidine phenyl drugs have emerged as promising candidates. However, how such current inhibition takes place remains obscure from the molecular point of view, hindering any further progress in developing drugs. Therefore, the nature of the binding sites, and how they are reached by the amidine-guanidine drugs, was investigated here via automated docking and molecular dynamics with hASIC1a homology models. This study has revealed that this ion channel is rich in binding sites, and that flexible drugs, such as nafamostat, may penetrate it in a snake-like elongated conformation. Then, crawling like a snake through temporary holes in the protein, nafamostat either simply flips, or changes to a high-energy folded conformation to become adapted to the shape of the binding site.     

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.     

Computational elucidation,mutational and hot spot-based designing of potential inhibitors against human acid-sensing ion channels (hASIC-1a) to treat various physiological conditions

Acid-sensing ion channels are ligand/proton-gated ion channels belonging to the family of the degenerin/epithelial Na⁺ channel (DEG/ENaC). They function as a sodium-selective pore for Ca²⁺ entry into neuronal cells during pathological conditions. The blocking of this channel has therapeutic importance, because at basal physiological pH (7.2), it is in a closed state and under a more acidic condition, and the ASIC1a ion channel is activated. To investigate the different states of the hASIC1a channel based on mutational analysis, structure-based virtual screening and molecular dynamics simulation studies. The system showed stability after 30 ns (after 1500 frame), and it was stabilized to an average value around 2.2Å. During the simulation, the ion channel residues in persistent contact with toxin PcTx1 were D237, E238, D347, D351, E219 and E355. These residues are important physiologically for the activation of the channel. From in silico alanine scanning, the significant hotspots obtained in hASIC1 are E344, P347, F352, D351, E355 and E219. From the sitemap analysis, it was evident that the sitemap found one of the active sites at the PcTx1 binding site with a site score of 1.086 and a D-score of 1.035 for hASIC1. We obtained a few promising hits and final potential hits from the virtual screening in hASIC1 that made interactions with the residues in the acidic pocket (E344, P347, F352, D351, E355 and E219). Based on these studies, the hits and scaffolds of potential therapeutic interest against various pathological conditions are associated with hASIC1a for future studies.     

Molecular Cloning and Regional Distribution of a Human Proton Receptor Subunit with Biphasic Functional Properties

Abstract : Small changes of extracellular pH activate depolarizing inward currents in most nociceptive neurons. It has been recently proposed that acid sensitivity of sensory as well as central neurons is mediated by a family of proton-gated cation channels structurally related to Caenorhabditis elegans degenerins and mammalian epithelial sodium channels. We describe here the molecular cloning of a novel human proton receptor, hASIC3, a 531-amino acid-long subunit homologous to rat DRASIC. Expression of homomeric hASIC3 channels in Xenopus oocytes generated biphasic inward currents elicited at pH <5, providing the first functional evidence of a human proton-gated ion channel. Contrary to the DRASIC current phenotype, the fast desensitizing early component and the slow sustained late component differed both by their cationic selectivity and by their response to the antagonist amiloride, but not by their pH sensitivity (pH₅₀ = 3.66 vs. 3.82). Using RT-PCR and mRNA blot hybridization, we detected hASIC3 mRNA in sensory ganglia, brain, and many internal tissues including lung and testis, so hASIC3 gene expression was not restricted to peripheral sensory neurons. These functional and anatomical data strongly suggest that hASIC3 plays a major role in persistent proton-induced currents occurring in physiological and pathological conditions of pH changes, likely through a tissue-specific heteropolymerization with other members of the proton-gated channel family.     

Reversibility of the Ca(2+) channel alpha(1)-beta subunit interaction

The auxiliary beta subunit importantly regulates voltage-dependent Ca(2+) channel activity through an interaction with the AID domain, a binding site located in the cytoplasmic I-II linker of the ion-conducting alpha(1) subunit. In the present study, we used two synthetic peptides corresponding to partial sequences of the I-II linker of alpha(1A) (AID(A)-peptides) as tools to disrupt the alpha(1)-beta interaction. In vitro binding experiments confirmed that these peptides exhibit a reasonable affinity to the neuronal beta(3) subunit to serve this purpose, although they failed to prevent immunoprecipitation of native N- and P/Q-type channels by anti-beta(3) antibodies. Together, our results (i) provide evidence for the reversibility of channel subunit association suggesting that the disruption of the alpha(1)-beta interaction may be a possible mechanism for Ca(2+) channel regulation in vivo, and (ii) support a model whereby the alpha(1)-beta association is based on multiple interaction sites.     

Potentiation of acid-sensing ion channels by sulfhydryl compounds

The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H⁺-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH_0.5 from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H⁺-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids. glutathione; DTT; redox; zinc     

A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels

Proton-gated ion channels conduct mainly Na⁺ to induce postsynaptic membrane depolarization. Finding the determinants of ion selectivity requires knowledge of the pore structure in the open conformation, but such information is not yet available. Here, the open conformation of the hASIC1a channel was computationally modeled, and functional effects of pore mutations were analyzed in light of the predicted structures. The open pore structure shows two constrictions of similar diameter formed by the backbone of the GAS belt and, right beneath it, by the side chains of H28 from the reentrant loop. Models of nonselective mutant channels, but not those that maintain ion selectivity, predict enlargement of the GAS belt, suggesting that this motif is quite flexible and that the loss of stabilizing interactions in the central pore leads to changes in size/shape of the belt. Our results are consistent with the “close-fit” mechanism governing selectivity of hASIC1a, wherein the backbone of the GAS substitutes at least part of the hydration shell of a permeant ion to enable crossing the pore constriction.     

From the Sequence to the Conformation of the Unabridged Transmembrane Domains TM1 and TM2 of the cASIC1a Ion Channel – A Parallel Tempering Approach

This work was devised to unravel, along replica‐exchange molecular‐dynamics (REMD) simulations, the conformation in solution of the TM1 and TM2 transmembrane domains of the homotrimeric cASIC1a ion channel. This includes the head of TM1 and tail of TM2 that had previously defied X‐ray diffraction analysis in the crystal. The structure of the open‐channel complex of cASIC1a with psalmotoxin 1 (PcTx1) was chosen here as a basis, although, to make the simulations affordable, the procedure was limited to the missing portions, including a few adjacent α‐helical turns. The latter were held fixed during the simulations. Reassembling the whole subunit, by superimposition of the fixed portions, resulted in diving of both TM1 and TM2 as continuous α‐helices into the cytoplasm. At completion of this work, it appeared, from similar X‐ray diffraction studies, that TM2 for both the complex of cASIC1a with the coral snake MitTx toxin, and the isolated desensitized ion channel, is discontinuous, with the triad G443‐A444‐S445 taking an extended, belt‐like conformation. In this way, a filter ring against hydrated ions is formed by G443 in the trimer. Our REMD examination of this complex revealed a strong resistance by G443, and only that residue, to take dihedral‐angle values compatible with an α‐helical conformation. This suggests that the flexibility of glycine alone does not explain formation of the extended, belt‐like conformation of the triad G443‐A444‐S445. This also requires cooperation in the trimer.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.     

Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azidopine with the calcium-channel alpha 1 subunit.

The dihydropyridine binding site of the rabbit skeletal muscle calcium channel alpha 1 subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrenidpine at an alpha 1 subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the alpha 1 subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp-N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13-18 (minor peak) and 1428-1437 (major peak) of the primary sequence of the skeletal muscle alpha 1 subunit. The nitrendipine peptides were identical with amino acids 1390-1399 (major peak) and 1410-1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the alpha 1 subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.     

Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.     

Identification of the benzothiazepine-binding polypeptide of skeletal muscle calcium channels with (+)-cis-azidodiltiazem and anti-ligand antibodies

The purified dihydropyridine-sensitive calcium channel from skeletal muscle transverse tubules consists of several subunits, termed alpha 1, alpha 2, beta, gamma and delta. From its associated drug receptors, those for 1,4-dihydropyridines and phenylalkylamines have been shown previously by photoaffinity labeling to reside on the alpha 1 subunit. In the present study the arylazide photo-affinity ligand, (+)-cis-azidodiltiazem ((+)-cis-(2S,3S)-5-[2-(4- azidobenzoyl)aminoethyl]-2,3,4,5-tetrahydro-3-hydroxy-2-(4-methoxyphenyl )-4- oxo-1,5-benzothiazepine), and the respective tritiated derivative, (+)-cis-[3H]azidodiltiazem (45 Ci/mmol), were developed to identify directly the benzothiazepine binding subunit. (+)-cis-Azidodiltiazem binds competitively to the benzothiazepine receptor in rabbit skeletal muscle transverse tubule membranes. Upon ultraviolet irradiation of the (+)-cis-[3H]azidodiltiazem-purified calcium channel complex, the ligand photoincorporates exclusively into the alpha 1 subunit. Photoincorporation is protected by 100 microM (-)-desmethoxyverapamil and 100 microM (+)-cis-diltiazem. A polyclonal antiserum directed against (+)-cis-azidodiltiazem was employed to detect (+)-cis-azidodiltiazem immunoreactivity photoincorporated into the purified calcium channel complex, confirming the exclusive labeling of the alpha 1 subunit. Our data provide direct evidence that, together with the drug receptors for 1,4-dihydropyridines and phenylalkylamines, the benzothiazepine binding domain of skeletal muscle calcium channels is located on the alpha 1 subunit. We conclude that our anti-ligand antibodies could be used successfully to affinity purify the photolabeled proteolytic fragments of the alpha 1 subunit which are expected to form part of the benzothiazepine binding domain.     

An intersubunit hydrogen bond in the nicotinic acetylcholine receptor that contributes to channel gating

The muscle nicotinic acetylcholine receptor is a large, allosteric, ligand-gated ion channel with the subunit composition alpha2betagammadelta. Although much is now known about the structure of the binding site, relatively little is understood about how the binding event is communicated to the channel gate, causing the pore to open. Here we identify a key hydrogen bond near the binding site that is involved in the gating pathway. Using mutant cycle analysis with the novel unnatural residue alpha-hydroxyserine, we find that the backbone N-H of alphaSer-191 in loop C makes a hydrogen bond to an anionic side chain of the complementary subunit upon agonist binding. However, the anionic partner is not the glutamate predicted by the crystal structures of the homologous acetylcholine-binding protein. Instead, the hydrogen-bonding partner is the extensively researched aspartate gammaAsp-174/deltaAsp-180, which had originally been identified as a key binding residue for cationic agonists.     

Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene

Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel.     

An arginine residue in the outer segment of hASIC1a TM1 affects both proton affinity and channel desensitization

Acid-sensing ion channels (ASICs) respond to changes in pH in the central and peripheral nervous systems and participate in synaptic plasticity and pain perception. Understanding the proton-mediated gating mechanism remains elusive despite the of their structures in various conformational states. We report here that R64, an arginine located in the outer segment of the first transmembrane domain of all three isoforms of mammalian ASICs, markedly impacts the apparent proton affinity of activation and the degree of desensitization from the open and preopen states. Rosetta calculations of free energy changes predict that substitutions of R64 in hASIC1a by aromatic residues destabilize the closed conformation while stabilizing the open conformation. Accordingly, F64 enhances the efficacy of proton-mediated gating of hASIC1a, which increases the apparent pH₅₀ and facilitates channel opening when only one or two subunits are activated. F64 also lengthens the duration of opening events, thus keeping channels open for extended periods of time and diminishing low pH-induced desensitization. Our results indicate that activation of a proton sensor(s) with pH₅₀ equal to or greater than pH 7.2–7.1 opens F64hASIC1a, whereas it induces steady-state desensitization in wildtype channels due to the high energy of activation imposed by R64, which prevents opening of the pore. Together, these findings suggest that activation of a high-affinity proton-sensor(s) and a common gating mechanism may mediate the processes of activation and steady-state desensitization of hASIC1a.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.     

KCNE1 binds to the KCNQ1 pore to regulate potassium channel activity

Potassium channels control the resting membrane potential and excitability of biological tissues. Many voltage-gated potassium channels are controlled through interactions with accessory subunits of the KCNE family through mechanisms still not known. Gating of mammalian channel KCNQ1 is dramatically regulated by KCNE subunits. We have found that multiple segments of the channel pore structure bind to the accessory protein KCNE1. The sites that confer KCNE1 binding are necessary for the functional interaction, and all sites must be present in the channel together for proper regulation by the accessory subunit. Specific gating control is localized to a single site of interaction between the ion channel and accessory subunit. Thus, direct physical interaction with the ion channel pore is the basis of KCNE1 regulation of K+ channels.