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Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

Nicotinic acetylcholine receptors (nAChRs) are members of the “cys-loop” ligand-gated ion channel superfamily that play important roles in both the peripheral and central system. At the neuromuscular junction, the endplate current is induced by ACh binding and nAChR activation, and then, the current declines to a small steady state, even though ACh is still bound to the receptors. The kinetics of nAChRs with high affinity for ACh but no measurable ion conductance is called desensitization. This adopted desensitization of nAChR channel currents might be an important mechanism for protecting cells against uncontrolled excitation. This study aimed to show that Grammostola spatulata toxin (GsMTx4), which was first purified and characterized from the venom of the tarantula Grammostola spatulata (now genus Phixotricus), can facilitate the desensitization of nAChRs in murine C2C12 myotubes. To examine the details, muscle-type nAChRs, which are expressed heterologously in HEK293T cells, were studied. A single channel current was recorded under the cell-attached configuration, and the channel activity (NPo) decayed much faster after the addition of GsMTx-4 to the pipette solution. The channel kinetics were further analyzed, and GsMTx-4 affected the channel activity of nAChRs by prolonging the closing time without affecting channel conductance or opening activity. The interaction between nAChRs embedded in the lipid membrane and toxin inserted into the membrane may contribute to the conformational change in the receptor and thus change the channel activity. This new property of GsMTx-4 may lead to a better understanding of the desensitization of ligand-gated channels and disease therapy.

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, &alpha, hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the ‘resting’ and ‘activated’ structures suggest a mechanism for ligand discrimination., eLife digest Ion channels are essential for transmitting signals in the nervous system and brain. One large group of ion channels includes members that are activated by cyclic nucleotides, small molecules used to transmit signals within cells. These cyclic nucleotide-gated channels play an important role in regulating our ability to see and smell. The activity of these ion channels has been studied for years, but scientists have only recently been able to look into their structure. Since structural biology methods require purified, well-behaved proteins, the members of this ion channel family selected for structural studies do not necessarily match those whose activity has been well established. There is a need for a good model that would allow both the structure and activity of a cyclic nucleotide-gated ion channel to be characterized. The cyclic nucleotide-gated ion channel, SthK, from bacteria called Spirochaeta thermophila, was identified as such model because both its activity and its structure are accessible. Rheinberger et al. have used cryo electron microscopy to solve several high-resolution structures of SthK channels. In two of the structures, SthK was bound to either one of two types of activating cyclic nucleotides – cAMP or cGMP – and in another structure, no cyclic nucleotides were bound. Separately recording the activity of individual channels allowed the activity states likely to be represented by these structures to be identified. Combining the results of the experiments revealed no activity from channels in an unbound state, low levels of activity for channels bound to cGMP, and moderate activity for channels bound to cAMP. Rheinberger et al. show that the channel, under the conditions experienced in cryo electron microscopy, is closed in all of the states studied. Unexpectedly, the binding of cyclic nucleotides produced no structural change even in the cyclic nucleotide-binding pocket of the channel, a region that was previously observed to undergo such changes when this region alone was crystallized. Rheinberger et al. deduce from this that the four subunits that make up the channel likely undergo the conformational change towards an open state all at once, rather than one by one. The structures and the basic functional characterization of SthK channels provide a strong starting point for future research into determining the entire opening and closing cycle for a cyclic nucleotide-gated channel. Human equivalents of the channel are likely to work in similar ways. The results presented by Rheinberger et al. could therefore be built upon to help address diseases that result from deficiencies in cyclic nucleotide-gated channels, such as loss of vision due to retinal degradation (retinitis pigmentosa or progressive cone dystrophy) and achromatopsia.

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na+:Ca2+,K+ exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K+, Na+ or Ca2+), the OS membrane resistance (R m ) was typically larger than 1 GΩ in the presence of 1 mM external Ca2+. Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in ∼50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

The properties of the pore of cyclic-nucleotide-gated channels are thought to depend mostly on the structure of the "P loop" which connects the S5 and S6 transmembrane segments. We applied scanning cysteine mutagenesis to the P loop of the alpha subunit of cGMP-gated channels from bovine rod, i.e. to the amino acid residues K346 to S371 (here named K2-S27). Mutant channels were expressed in Xenopus laevis oocytes and currents recorded from inside-out patches, in symmetrical sodium and in the absence of divalent cations. Cysteine mutations had minor effects on the affinity for cGMP and the selectivity to monovalent alkali cations. However, in mutants within the segment W9-T16, open probability was strongly reduced. It was less than 0.2 in saturating cGMP compared to 0.8 in wild-type channels. Moreover, W9C and L12C mutants were outward rectifiers, while T16C was an inward rectifier. These mutant channels showed a time dependence in the development of the steady-state current. Currents from I17C channels progressively decayed in inside-out patches. This rundown was prevented by 1 mM dithiothreitol on the cytoplasmic side of the plasma membrane, suggesting that I17 is an intracellular residue. On the other hand, mutants within the segment T20-S27 had normal open probabilities, around 0.8 in saturating cGMP, but altered single-channel conductance.

Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+-activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+(Ca2+) channels with a Kd of 1–3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.