Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.     

Permeation and block by internal and external divalent cations of the catfish cone photoreceptor cGMP-gated channel

The ability of the divalent cations calcium, magnesium, and barium to permeate through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions and by measuring their ability to block current carried by sodium when presented on the cytoplasmic or extracellular side of the channel. Current carried by divalent cations in the absence of monovalent cations showed the typical rectification pattern observed from these channels under physiological conditions (an exponential increase in current at both positive and negative voltages). With calcium as the reference ion, the relative permeabilities were Ca > Ba > Mg, and the chord conductance ratios at +50 mV were in the order of Ca approximately Mg > Ba. With external sodium as the reference ion, the relative permeabilities were Ca > Mg > Ba > Na with chord conductance ratios at +30 mV in the order of Na >> Ca = Mg > Ba. The ability of divalent cations presented on the intracellular side to block the sodium current was in the order Ca > Mg > Ba at +30 mV and Ca > Ba > Mg at -30 mV. Block by external divalent cations was also investigated. The current-voltage relations showed block by internal divalent cations reveal no anomalous mole fraction behavior, suggesting little ion-ion interaction within the pore. An Eyring rate theory model with two barriers and a single binding site is sufficient to explain both these observations and those for monovalent cations, predicting a single-channel conductance under physiological conditions of 2 pS and an inward current at -30 mV carried by 82% Na, 5% Mg, and 13% Ca.     

Interaction between calcium ions and surface charge as it relates to calcium currents

Summary Calcium ions affect the gating of Ca currents. Surface charge is involved but to what extent is unknown. We have examined this, using isolated nerve cell bodies ofHelix aspersa and the combined microelectrode-suction pipette method for voltage-clamp and internal perfusion. We found that Ba and Sr currents produced by substitution of these ions for extracellular Ca ions are activated at less positive potentials than Ca currents. Mg ions do not permeate the Ca channel and changes in [Mg]₀ produce shifts in the activation-potential curves that are comparable to the effects of changes in [Ba]₀ or [Sr]₀. Inactivation of Ba currents also occurs at less positive potentials. Perfusion intracellularly with EGTA reduced inactivation of Ca currents as a function of potential, but did not shift the inactivation-potential curve. Hence, Ca current-dependent inactivation which is blocked by intracellular EGTA probably does not involve a similar change of intracellular surface potential. The voltage shifts of activation and inactivation produced by extracellular divalent cations used singly or in mixtures can be described by the Gouy-Chapman theory for the diffuse double layer with binding (Gilbert & Ehrenstein, 1969; McLaughlin, Szabo & Eisenman, 1971). From the surface potential values and the Boltzman distribution, we have computed surface concentrations that predict the following experimental observations: 1) saturation of current-concentration relationships when surface potential is changing maximally; 2) the increase in peak current when Ca ions are replaced by Sr or Ba ions; and 3) the greater inhibitory effect of Mg onI _Ba thanI _Ca. Theory indicates that surface charge cannot be screened completely even at 1m [Mg]₀ and thus that Ca channel properties must be evaluated in the light of surface charge effects. For example, after correction for surface charge effects the relative permeabilities of Ca, Ba and Sr ions are equivalent. In the presence of Co ions, however, Ca ions are more permeable than Ba ions suggesting a channel binding site may be involved.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Effects of divalent cations on toad end-plate channels

Summary Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltageclamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20mm Ca or Mg, MEPCs had time constants of decay ( _D ) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both _D and channel lifetime were seen; the null potential was –34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1–5mm, were much more effective in increasing _D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in , especially when taken together with changes in channel conductance.     

Monovalent currents through the T-type Cav3.1 channels and their block by Mg2+

We have investigated the permeability of the Cav3.1 channel for Ca2+ and different monovalent cations and the block of the currents by Mg2+ ions. In the absence of extracellular divalent cations, the Cav3.1 channel was more permeable for Na+ than for Cs+ and impermeable for NMDG+. Monovalent currents were inhibited by Mg2+ of near physiological concentration by three orders of magnitude more effectively than the Ca2+ current. Inhibition of outward, but not inward current by Mg2+ was voltage-dependent. Furthermore, magnesium slowed down channel deactivation presumably by interacting with an open channel state.     

Ionic permeability characteristics of the N-methyl-D-aspartate receptor channel

N-methyl-D-aspartate (NMDA) receptor channels in cultured CA1 hippocampal neurons were studied using patch-clamp techniques. The purpose of the research was to determine the occupancy of the channel by permeant cations and to determine the influence of charged residues in or near the pore. The concentration dependence of permeability ratios, the mole-fraction dependence of permeability ratios, the concentration dependence of the single-channel conductance, and a single-channel analysis of Mg2+ block all independently indicated that the NMDA receptor behaves as a singly-occupied channel. More precisely, there is one permeant cation at a time occupying the site or sites that are in the narrow region of the pore directly in the permeation pathway. Permeability-ratio measurements in mixtures of monovalent and divalent cations indicated that local charges in or near the pore do not produce a large local surface potential in physiologic solutions. In low ionic strength solutions, a local negative surface potential does influence the ionic environment near the pore, but in normal physiologic solutions the surface potential appears too small to significantly influence ion permeation. The results indicate that the mechanism for the high Ca2+ conductance of the NMDA receptor channel is not the same as for the voltage-dependent Ca2+ channel (VDCC). The VDCC has two high affinity, interacting binding sites that provide high Ca2+ selectivity and conductance. The binding site of the NMDA receptor is of lower affinity. Therefore, the selectivity for Ca2+ is not as high, but the lower affinity of binding provides a faster off rate so that interacting sites are not required for high conductance.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel

Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.     

Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells

Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.     

Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity

Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective alpha1 homomeric glycine receptor (alpha1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1'E GlyR) was sufficient to convert the channels to select cations over anions with P(Cl)/P(Na) = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2'Delta mutant GlyR retained its anion selectivity (P(Cl)/P(Na) = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (P(Cl)/P(Na) = 27.9). When the A-1'E and the P-2'Delta mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (P(Cl)/P(Na) = 0.13). The SDM GlyR was also Ca(2+) permeable (P(Ca)/P(Na) = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19'A GlyR) produced a more Ca(2+)-permeable channel (P(Ca)/P(Na) = 0.73), without drastically altering monovalent charge selectivity (P(Cl)/P(Na) = 0.23). The SDM+R19'E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca(2+) permeability (P(Ca)/P(Na) = 0.92), with little effect on monovalent selectivity (P(Cl)/P(Na) = 0.19). Estimates of the minimum pore diameter of the A-1'E, SDM, SDM+R19'A, and SDM+R19'E GlyRs revealed that these pores are larger than the alpha1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Divalent effects on cGMP-activated currents in excised patches from amphibian photoreceptors

Summary The light-sensitive current in photoreceptors is conducted by a single class of ion channels gated by the binding of multiple molecules of cytoplasmic cGMP. Both Na and Ca ions enter the outer segment through this channel and Ca behaves as a blocking ion, greatly reducing the influx of Na. Because intracellular Ca functions as the cytosolic messenger for light adaptation, and this channel is the major entry point for Ca into the outer segment, we seek a better understanding of the selectivity properties of the channel and how they affect intracellular Ca levels. In these studies, we added divalent cations to the cytoplasmic face of an excised patch at constant, symmetrical [Na]. Our results suggest a novel high-affinity divalent binding site at the internal face of the channel. At constant low levels of cGMP, the addition of 10–100nm cytoplasmic Ca or Mg attenuated the current 5- to 10-fold. There is also a low-affinity site, midway through the transmembrane field; saturation of this site reduces the divalent-free current 100-fold. The presence of a high-affinity cytoplasmic site raises the question of whether Ca regulates the photoreceptor current through a direct interaction with the channel perhaps altering the channel selectivity or kinetics.     

Ion selectivity of epithelial Na channels

Epithelial Na channels are apparently pore-forming membrane proteins which conduct Na much better than any other biologically abundant ion. The conductance to Na can be 100 to 1000 times higher than that to K. The only other ions that can readily get through this channel are protons and Li. Small organic cations cannot pass through the channel, and water may also be impermeant. The selectivity properties of epithelial Na channels appear to be determined by at least three factors: A high field-strength anionic site, most likely a carboxyl residue of glutamic or aspartic acid residues on the channel protein, probably accounts for the high conductance through these channels of Na and Li and to the low conductance of K, Rb and Cs. A restriction in the size of the pore at its narrowest point probably accounts for the low conductance of organic cations as well as the possible exclusion of water molecules. The outer mouth of the channel appears to be negatively charged and may control access to the region of highest selectivity and may serve as a preliminary selectivity filter, attracting cations over anions. These conclusions are illustrated by the cartoon of the channel in Fig. 3. This picture is obviously both fanciful and simplified, but its general points will hopefully be testable. It leaves open a number of important questions, including: does amiloride block the channel by binding within the outer mouth? what does the inner mouth of the channel look like, and does this part of the channel contribute to selectivity? and what, if any, are the interactions between the features of the channel that impart selectivity and those that control the regulation of the channel by hormonal and other factors?     

Conduction of Monovalent and Divalent Cations in the Slow Vacuolar Channel

The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.     

Monovalent ion current through single calcium channels of skeletal muscle transverse tubules.

Akali monovalents, Li, Na, K, Cs, and organic monovalents of molecular cross section less than 20 A2, ammonium, methylammonium, hydrazinium, guanidinium, are shown to have a measurable conductance through Ca channels of muscle transverse tubules reconstituted into planar bilayers. For the alkali series, single channel conductances follow the sequence Cs approximately equal to K greater than Na greater than Li with a conductance ratio [g(Cs)/g(Li)] = 1.7. For permeability ratios, the sequence is Li greater than Na greater than K approximately equal to Cs with [P(Li)/P(Cs)] = 1.5. Monovalent current is only unmasked when Ba ions are not present. In mixtures of Cs and Ba, single channel current reverses close to the Ba equilibrium potential and more than 100 mV away from the Cs equilibrium potential. A cutoff in conduction is found for organic cations larger than trimethylammonium; this suggests an apparent pore aperture of about 5 X 5 A. Even in such a large pore, the fact that the alkali cation permeability sequence and conductance sequence are inverted rules out molecular sieving as the mechanism of selection among monovalents.     

Apical membrane K conductance in the toad urinary bladder

Summary The conductance of the apical membrane of the toad urinary bladder was studied under voltage-clamp conditions at hyperpolarizing potentials (mucosa negative to serosa). The serosal medium contained high KCl concentrations to reduce the voltage and electrical resistance across the basal-lateral membrane, and the mucosal solution was Na free, or contained amiloride, to eliminate the conductance of the apical Na channels. As the mucosal potential (V _m) was made more negative the slope conductance of the epithelium increased, reaching a maximum at conductance of the epithelium increased, reaching a maximum atV _m=–100 mV. This rectifying conductance activated with a time constant of 2 msec whenV _m was changed abruptly from 0 to –100 mV, and remained elevated for at least 10 min, although some decrease of current was observed. ReturningV _m to+100 mV deactivated the conductance within 1 msec. Ion substitution experiments showed that the rectified current was carried mostly by cations moving from cell to mucosa. Measurement of K flux showed that the current could be accounted for by net movement of K across the apical membrane, implying a voltage-dependent conductance to K (G _K). Mucosal addition of the K channel blockers TEA and Cs had no effect onG _K, while 29mm Ba diminished it slightly. Mucosal Mg (29mm) also reducedG _K, while Ca (29mm) stimulated it.G _K was blocked by lowering the mucosal pH with an apparent pK₁ of 4.5. Quinidine (0.5mm in the serosal bath) reducedG _K by 80%.G _K was stimulated by ADH (20 mU/ml), 8-Br-cAMP (1mm), carbachol (100 m), aldosterone (5×10^–7 m for 18 hr), intracellular Li and extracellular CO₂.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.     

Changes in Negative Charge at the Luminal Mouth of the Pore Alter Ion Handling and Gating in the Cardiac Ryanodine-Receptor

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K⁺ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.