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Molecular modeling and simulations enable extrapolation for the structure of bacterial potassium channels to the function of their mammalian homologues. Molecular dynamics simulations have revealed the concerted single-file motion of potassium ions and water molecules through the selectivity filter of K channels and the role of filter flexibility in ion permeation and in "fast gating." Principal components analysis of extended K channel simulations suggests that hinge-bending of pore-lining M2 (or S6) helices plays a key role in K channel gating. Based on these and other simulations, a molecular model for gating of inward rectifier K channel gating is presented. [ABSTRACT FROM PUBLISHER]

Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This paper will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which plays a critical role in the fast chemical transmission of electrical signals between nerve cells and between nerve and muscle cells. The superfamily will mainly be exemplified by the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels. [ABSTRACT FROM PUBLISHER]

Gramicidin channels are mini-proteins composed of two tryptophan-rich subunits. The conducting channels are formed by the transbilayer dimerization of nonconducting subunits, which are tied to the bilayer/solution interface through hydrogen bonds between the indole NH groups and the phospholipid backbone and water. The channel structure is known at atomic resolution and the channel's permeability characteristics are particularly well defined: gramicidin channels are selective for monovalent cations, with no measurable permeability to anions or polyvalent cations; ions and water move through a pore whose wall is formed by the peptide backbone; and the single-channel conductance and cation selectivity vary when the amino acid sequence is varied, even though the permeating ions make no contact with the amino acid side chains. Given the amount of experimental information that is available-for both the wild-type channels and for channels formed by amino acid-substituted gramicidin analogues-gramicidin channels provide important insights into the microphysics of ion permeation through bilayer-spanning channels. For the same reason, gramicidin channels constitute the system of choice for evaluating computational strategies for obtaining mechanistic insights into ion permeation through the complex channels formed by integral membrane proteins. [ABSTRACT FROM PUBLISHER]

Bestrophins have been proposed to constitute a new family of Cl channels that are activated by cytosolic Ca. We showed previously that mutation of serine-79 to cysteine in mouse bestrophin-2 (mBest2) altered the relative permeability and conductance to SCN. In this paper, we have overexpressed various mutant constructs of mBest2 in HEK-293 cells to explore the contributions to anion selectivity of serine-79 and other amino acids (V78, F80, G83, F84, V86, and T87) located in the putative second transmembrane domain (TMD2). Residues selected for mutagenesis were distributed throughout TMD2, but mutations at all positions changed the selectivity The effects on selectivity were rather modest. Replacement of residues 78, 79, 80, 83, 84, 86, or 87 with cysteine had similar effects: the permeability of the channel to SCN relative to Cl (PSCN/PCl) was decreased three- to fourfold and the relative SCN conductance (GSCN/GCl) was increased five- to tenfold. Side chains at positions 78 and 80 appeared to be situated close to the permeant anion, because the electrostatic charge at these positions affected permeation in specific ways. The effects of charged sulfhydryl-reactive MTS reagents were the opposite in the V78C and F8OC mutants and the effects were partially mimicked by substitution of F80 with charged amino acids. In S79T, switching from Cl to SCN caused slow changes in GSCN/GCl (τ = 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked. [ABSTRACT FROM AUTHOR]

Tetrodotoxin-resistant (TTX-R) Na+ channels are much less susceptible to external TTX but more susceptible to external Cd2+ block than tetrodotoxin-sensitive (TTX-S) Na+ channels. Both TTX and Cd2+ seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La3+, Zn2+, Ni2+, Co2+, and Mn2+ also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd2+, the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na+ flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na+ currents than those in outward Na+ current, signaling exit of the blocker along with the Na+ flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na+ current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na+ flow-dependent unblocking of the bound La3+ from the open Na+ channel, whereas channel inactivation cannot happen with any discernible speed in the La3+-blocked channel. Thus, the selectivity filter of Na+ channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel. [ABSTRACT FROM AUTHOR]

A three-state hopping model is established according to experiments to study permeation of an open-state potassium channel from Streptomyces Lividans (KcsA potassium channel). The master equations are used to characterize the dynamics of the system. In this model, ion conduction involves transitions of three states, with one three-ion state and two two-ion states in the selectivity filter respectively. In equilibrium, the well-known Nernst equation is deduced. It is further shown that the current follows Michaelis-Menten kinetics in steady state. According to the parameters provided by Nelson, the current-voltage relationship is proved to be ohmic and the current-concentration relationship is also obtained reasonably. Additional validation of the model in the characteristic time to reach the steady state for the potassium channel is also discussed. This model lays a possible physical basis for the permeation of ion channel, and opens an avenue for further research. [ABSTRACT FROM AUTHOR]

Bestrophins have recently been proposed to comprise a new family of Cl- channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl- channel. We expressed mBest2 in three different mammalian cell lines, mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl- current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN-: I-: Br-: Cl-: F- (8.2: 1.9: 1.4: 1: 0.5). Although SGN- was highly permeant, its conductance was ∼10% that of Cl- and SCN- blocked Cl- conductance (IC50 = 12 mM). Therefore, SCN entered the pore more easily than Cl-, but bound more tightly than Cl-. Mutations in S79 altered the relative permeability and conductance for SCN- as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN- did not block Cl- conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN-. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES- increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl- conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel. [ABSTRACT FROM AUTHOR]

Low voltage-activated (LVA) Ca2+ channels regulate chemical signaling by their ability to select for Ca2+. Whereas Ca2+ is the main permeating species through Ca2+ channels, Ca2+ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca2+ permeation in the cloned T-type Ca2+ channels α1G (CaV3.1) and α1H (CaV3.2). In physiological [Ca2+], the reversal potential in symmetrical Li+ was 19 mV in α1G and 18 mV in α1H, in symmetrical Cs+ the reversal potential was 36 mV in α1G and 37 mV in α1H, and in the bi-ionic condition with Li+ in the bath and Cs+ in the pipette, the reversal potential was 46 mV in both α1G and α1H. When Cs+ was used in the pipette, replacement of external Cs+ with Li+ (or Na+) shifted the reversal potential positive by 5–6 mV and increased the net inward current in α1G. Taken together the data indicate that in physiological [Ca2+], external Li+ (or Na+) permeates more readily than external Cs+, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca2+ channel selectivity by permeating through the channel. Similar to Li+, we previously reported that external [H+] can regulate T-type Ca2+ channel selectivity. α1H’s selectivity was more sensitive to external pH changes compared to α1G. When Cs+ was used in the pipette and Li+ was used in the bath external acidification from pHo 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in α1H. Replacement of internal Cs+ with Li+ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H+ can modify T-type Ca2+ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H+ regulate T-type Ca2+ channel selectivity by increasing the relative permeability of the internal monovalent cation. [ABSTRACT FROM AUTHOR]

α7 nicotinic receptors are highly permeable to Ca2+ as well as monovalent cations. We extended the characterization of the Ca2+ permeation of non-desensitizing chick α7 receptors (S240T/L247T α7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca2+ or Ba2+ were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca2+ block of K+ currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca2+ activity (2.4 mM concentration) for Ca2+ permeation and an apparent affinity of 0.65 mM Ba2+ activity (1.7 mM concentration) for Ba2+ permeation. The apparent affinity of Ca2+ inhibition of K+ currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of α7 receptors has one or more low-affinity Ca2+ binding sites and no high-affinity sites. [Copyright &y& Elsevier]

Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ∼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore. [Display omitted] • Exocytotic transmitter release is an electrodiffusion process • Narrow fusion pores are cation selective • Fusion pore ion selectivity decreases as the fusion pore expands For transmission, a fusion pore forms when vesicle and target membranes are brought together by SNARE proteins. Delacruz et al. demonstrate that selectivity of the pore accelerates release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore. [ABSTRACT FROM AUTHOR]

[Display omitted] • K+ channels are characterized by the highly efficient and selective permeation of K+ ions. • Determining the ion permeation mechanism in K+ channels would explain their remarkable conductive properties. • Many experimental and computational studies have been performed, and multiple ion permeation mechanisms have been proposed - however, the controversies in the field persist and consensus on which mechanism actually occurs is yet to be reached. • In this review, we discuss important milestones in research of ion permeation mechanisms in K+ channels and compare the results obtained with different approaches, as well as present further challenges and possible solutions. Potassium channels play critical roles in many physiological processes, providing a selective permeation route for K+ ions in and out of a cell, by employing a carefully designed selectivity filter, evolutionarily conserved from viruses to mammals. The structure of the selectivity filter was determined at atomic resolution by x-ray crystallography, showing a tight coordination of desolvated K+ ions by the channel. However, the molecular mechanism of K+ ions permeation through potassium channels remains unclear, with structural, functional and computational studies often providing conflicting data and interpretations. In this review, we will present the proposed mechanisms, discuss their origins, and will critically assess them against all available data. General properties shared by all potassium channels are introduced first, followed by the introduction of two main mechanisms of ion permeation: soft and direct knock-on. Then, we will discuss critical computational and experimental studies that shaped the field. We will especially focus on molecular dynamics (MD) simulations, that provided mechanistic and energetic aspects of K+ permeation, but at the same time created long-standing controversies. Further challenges and possible solutions are presented as well. [ABSTRACT FROM AUTHOR]

Prokaryotic channels play an important role in the structural biology of ion channels. At the end of the 20 th century, the first structure of a prokaryotic ion channel was revealed. Subsequently, the reporting of structures of various prokaryotic ion channels have provided fundamental insights into the structure of ion channels of higher organisms. Voltage-dependent Ca 2+ channels (Cavs) are indispensable for coupling action potentials with Ca 2+ signaling. Similar to other proteins, Cavs were predicted to have a prokaryotic counterpart; however, it has taken more than 20 years for one to be identified. The homotetrameric channel obtained from Meiothermus ruber generates the calcium ion specific current, so it is named as CavMr. Its selectivity filter contains a smaller number of negatively charged residues than mutant Cavs generated from other prokaryotic channels. CavMr belonged to a different cluster of phylogenetic trees than canonical prokaryotic cation channels. The glycine residue of the CavMr selectivity filter is a determinant for calcium selectivity. This glycine residue is conserved among eukaryotic Cavs, suggesting that there is a universal mechanism for calcium selectivity. A family of homotetrameric channels has also been identified from eukaryotic unicellular algae, and the investigation of these channels can help to understand the mechanism for ion selection that is conserved from prokaryotes to eukaryotes., (2021 THE BIOPHYSICAL SOCIETY OF JAPAN.)

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wildtype current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation. [ABSTRACT FROM AUTHOR]

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wildtype current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation. [ABSTRACT FROM AUTHOR]

The mechanism of ion permeation in K/Na-permeable I channels of tiger salamander rod photoreceptors was investigated using the whole-cell voltage-clamp technique. I channels showed features indicative of pores with multiple ion binding sites: in mixtures of K and thallium Tl, the amplitude of the time-dependent current showed an anomalous mole fraction dependence, and K permeation was blocked by other permeant ions (with K values: Tl, 44 μM; Rb, 220 μM and NH, 1100 /gmM) as well as by essentially impermeant ions (Cs, 22 μM Ba, 9200 μM) which apparently block I by binding in the pore. In contrast, Na had little blocking action on K permeation. The block by all of these ions was sensitive to external K with the block by Cs being the least sensitive. Na was more effective than K in reducing the block by Tl, Rb and NH, but was less effective for the block by Cs and Ba. The blocking action of Cs and Ba was non-competitive, suggesting that they block I channels at independent sites. Based on the efficacy of block by the different ions, the degree to which K and Na antagonize this block and the noncompetitive blocking action of Cs and Ba, the permeation pathway of I channels appears to contain at least three ion binding sites with at least two sites having a higher affinity for K over Na and another site with a higher affinity for Na over K. [ABSTRACT FROM AUTHOR]

A conservative reversion at position 374 in a chimeric K pore, CHM, switched the preferred ionic conductance from K to Rb. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb-preferring pores while channels with the substituted polar residues Thr or Ser expressed K-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K and Rb ions and a site for blockade by internal TEA. [ABSTRACT FROM AUTHOR]

The kinetics of single K channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the 'tilt effect' model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092-8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter vacuolar or 1 mmol liter cytoplasmic Ca; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca at membrane PDs more negative than −100 mV; and (3) the effect of Mg on channel conductance when added to solutions initially containing 3 mmol liter KCl. The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than −70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg of surface potentials arising from diffusion-limited flow of K. All experimental approaches give a consistent picture of K permeation in which the surface charge and convergence permeability of the cytoplasmic vestibule are the major factors in determining channel conductance. The cytoplasmic vestibule has a charge density of −0.035 C/m which is similar to that found for maxi K channels in rat muscle. The properties of the vacuolar vestibule, which is effectively neutral, differ from the negatively charged external vestibules in rat maxi K channels indicating a differing protein structure in this part of the channel. Finally, we note that our method of testing for diffusion-limited ion flow, by measuring the dependence of the surface PD on the current passing through the channel, is more reliable than common tests, which make use of nonelectrolytes such as sucrose. It appears that these molecules alter channel conductance by interfering with the intrinsic permeation mechanism of the channel rather than by altering bulk viscosity. [ABSTRACT FROM AUTHOR]

The gigaohm seal technique was used to study ion permeation through acetylcholine-activated channels in cell-attached patches of the extrajunctional membrane of chronically denervated, enzyme-treated cells from the sartorius muscle of the toad Bufo marinus. The most frequently occurring channel type (greater than 95% of channel openings), provisionally classified as 'extrajunctional,' had a chord conductance of approximately 25 pS under normal conditions (-70 mV, 11 degrees C, Normal Toad Ringer's). The less frequently observed channel type (less than 5% of channel openings), classified as a 'junctional' type, had a conductance of 35 pS under the same conditions, and a similar null potential. In many patches, a small percentage (usually less than 2%) of openings of the extrajunctional channel displayed a lower conductance state. The shape of the I-V curves obtained for the extrajunctional channel depended on the predominant extracellular cation. For Cs and K, the I-V curves were essentially linear over the voltage range +50 to -150 mV across the patch, suggesting that the potential independent component of the energy profile within the channel was symmetrical. For Li, the I-V curve was very nonlinear, displaying a significant sublinearity at hyperpolarized potentials. Both an electrodiffusion and a symmetrical uniform four-barrier, three-site rate-theory model provided reasonable fits to the data, whereas symmetrical two-barrier, single-site rate-theory models did not. For the alkali cations examined, the relative permeability sequence was PCs greater than PK greater than PNa greater than PLi--a "proportional" selectivity sequence. This was different from the single channel conductance sequence which was found to be gamma K greater than gamma Cs greater than gamma Na greater than gamma Li implying that ions do not move independently through the channel. The relative binding constant sequence for the channel sites was found to be a "polarizability" sequence, i.e., KLi greater than KCs greater than KNa greater than KK. There was an inverse relationship between the relative binding constant and the relative mobility for the cations examined. Under conditions when the single-channel conductance was relatively high, the conductance at depolarized potentials was lower than that predicted by both electrodiffusion and rate theory models, suggesting that there was a rate-limiting access step for ions, from the intracellular compartment into the channel. [ABSTRACT FROM AUTHOR]

The members of the RCK family of cloned voltage-dependent K channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K and thereby indirectly affects internal Mg block and channel closing. [ABSTRACT FROM AUTHOR]

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L-α-lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl channel had the halide permeability sequence Cl > I ≥ Br > F, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl and F, ions. The autwardly rectifying Cl channel had the halide permeability sequence I > Br > Cl > F, and also showed anomalous molefraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed. [ABSTRACT FROM AUTHOR]

A functional model for the S4/IV α-helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the α-helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (φψ)=(87°, 315°) and (φψ)=(22°, 107°) which yields a helix of 13.5 Å per turn. The axial dipole moments of the peptide bonds of this chain of l-amino acids nearly cancel each other out in similar way to those in the gramicidin A channel, which is formed by alternating d-and l-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable 'double helix' structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the α-helix are broken and the internal α-carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the α-helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the α-helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an α-helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed. [ABSTRACT FROM AUTHOR]

In the lobster stretch receptor neurone it is possible to demonstrate a hyperpolarization-activated membrane current, I, which appears to be carried by Na and K in combination. The ion permeability of the membrane channel conducting this current ( Q-channel) was investigated using conventional electrophysiological techniques including intracellular ion concentration measurements. It was found that none of the ions choline, protonated Tris, Rb, NH, Li, and protonated hydroxylamine was able to pass through the Q-channel which, thus, appears to be permeable to Na and K only. With increasing extracellular Na concentrations, I was increased up to a saturation level. This behaviour could be described by a one-site-two-barriers version of the Eyring rate theory, assuming that the permeant ions are turned over at specific saturable channel sites which 'sense' 70% of the transmembrane potential difference. With increasing extracellular K concentrations, I was increased in accordance with a simple first-order doseresponse relationship. This finding can be accounted for by assuming that K increases all rates of turn-over of the permeant ions at their specific sites by similar relative amounts. Changes in extracellular Na and K concentrations were found to have no effect on the gating properties of the Q-channel. [ABSTRACT FROM AUTHOR]

The gigaohm seal technique was used to study the effects of temperature on ion permeation through acetylcholine-activated channels. This was done in cell-attached patches of the extrajunctional membrane of chronicallydenervated, enzyme-treated cells from sartorius muscle of the toad Bufo marinus. The predominant extracellular cation in the pipette solution was Na. Single channel currentvoltage curves were measured at different temperatures and electrodiffusion and three-site-four-barrier rate theory models were used to characterize ion permeation through the channels and determine the effects of temperature on permeation parameters. The fitting of the experimental data to these models suggested the presence of at least three and probably more ion-selective sites within the channel. The most frequently occuring channel type (>95% of channel openings) had a chord conductance of 25 pS at 11°C and −70 mV and was classified as 'extrajunctional'. The single channel conductance of this channel had a low temperature-dependence ( Q≈1.3). The apparent activation enthalpy, E, for the conductance between 11°C and 20°C, did not appear to be significantly voltage-sensitive and had a value of about 17±2 kJ·mol at a voltage of −70 mV. The Arrhenius plot of conductance appeared linear between 11 and 20°C at all potentials examined. The data was consistent with a break in the slope of the Arrhenius plot at temperatures between 5 and 11°C at all potentials examined, suggesting a possible phase transition of the membrane lipids. In contrast to the relative permeability, which was not very temperature sensitive, the relative binding constant was significantly affected by temperature. The relative Na/K binding constant sequence was: K> K> K≫ K. In addition, the decrease in conductance observed at the most depolarized potentials was accentuated as the temperature was increased, suggesting a rate-limiting access step for ions from the intracellular solution into the channel. [ABSTRACT FROM AUTHOR]

Diffusion potentials and conducatance measurements were used to evaluate the changes in permeability of the rabbit gall bladder when the lateral spaces were 1) closed by the addition of sucrose to the mucosal fluid, and 2) dilated by the addition of sucrose to the serosal fluid. The results showed that when the lateral spaces were closed (<10 nm/ 1) there was a significant decrease in the conductance of the epithelium, and 2) the ion selectivity of the epithelium moved towards the free solution sequence. The conductance decreased from 31 to 13 mmhos/cm, and the selectivety changed from Na(1)>Li(0.92)>Cs(0.85) to Cs(1.27)>Na(1)>Li(0.84). Neither dilation of the spaces to>1.5 μm nor addition of sucrose to both sides of the gall bladder changed the conductance or the ion selectivity. These results are consistant with the hypothesis that in the gall bladder the major barrier to ion permeation across the epithelium lies in 1) the tight junctions, when the lateral spaces are dilated, 2) the lateral spaces when the spaces are collapsed, and 3) a combination of both the spaces and the junctions when the spaces are reduced much below 0.5 μm Consequently the status of the lateral intercellular spaces has to be taken into avcount when assessing the mechanisms of ion permeation across low resistance epithelia. [ABSTRACT FROM AUTHOR]

Selective calcium channel antagonists are widely used in the treatment of cardiovascular disorders. They are mainly classified into 1,4-dihydropyridine (1,4-DHPs) and non-DHPs. The non-DHPs class is further classified into phenylalkylamines (PAAs) and benzothiazepines (BZTs) derivatives. These blockers are used for the treatment of hypertension, angina pectoris, and cardiac arrhythmias. Despite their well-established efficiency, the structural basis behind their activity is not very clear. Here we report the use of a near-open confirmation (NOC) model of the Cav1.2 cardiac ion channel to examine the mode of binding of these antagonists within the pore domain as well as the fenestration of the pore-forming domains. Effects of calcium ion permeation in the presence of drug molecules were assessed using steered molecular dynamics (SMD) simulations. These studies reveal that nicardipine, a DHP derivative, shows a strong Cav1.2 blocking activity, requiring more 2500 pN force to pull calcium ion towards the channel's pore in the presence of the compound. Similar blocking activity was observed for verapamil, a PAA derivative, requiring almost 2300 pN of force. The least blocking activity was observed for Diltiazem, a BZT derivative. Our results explain the structural basis and the binding details of 1,4-DHPs, PAAs and BZTs at their distinct Cav1.2 sites and offer detailed insights into their mechanism of action in modulating the Cav1.2 channel. Image 1 • Selective calcium channel blockers (CCBs) include 1,4-dihydropyridine (1,4-DHPs), phenylalkylamines (PAAs) and benzothiazepines (BZTs). • The structural basis behind CCBs' activity is not very clear. • Here we report the use of computational modelling to understand the modes of binding of various selective CCBs and their effects on ion permeation. • Our results explain the binding details of DHPs, PAAs and BZTs at their distinct Ca V 1.2 sites. • Our findings offer detailed insights into the mechanism of action of CCBs in modulating the Ca V 1.2 channel. [ABSTRACT FROM AUTHOR]