Cation permeation through the voltage-dependent potassium channel in the squid axon. Characteristics and mechanisms

Characteristics of cation permeation through voltage-dependent delayed rectifier K channels in squid giant axons were examined. Axial wire voltage-clamp measurements and internal perfusion were used to determine conductance and permeability properties. These K channels exhibit conductance saturation and decline with increases in symmetrical K+ concentrations to 3 M. They also produce ion- and concentration-dependent current-voltage shapes. K channel permeability ratios obtained with substitutions of internal Rb+ or NH+4 for K+ are higher than for external substitution of these ions. Furthermore, conductance and permeability ratios of NH+4 or Rb+ to K+ are functions of ion concentration. Conductance measurements also reveal the presence of an anomalous mole fraction effect for NH+4, Rb+, or Tl+ to K+. Finally, internal Cs+ blocks these K channels in a voltage-dependent manner, with relief of block by elevations in external K+ but not external NH+4 or Cs+. Energy profiles for K+, NH+4, Rb+, Tl+, and Cs+ incorporating three barriers and two ion-binding sites are fitted to the data. The profiles are asymmetric with respect to the center of the electric field, have different binding energies and electrical positions for each ion, and (for K+) exhibit concentration-dependent barrier positions.     

Further characterization of the cation channel of a yeast vacuolar membrane in a planar lipid bilayer

A voltage-dependent and Ca2(+)-activated cation channel recently found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH4+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determined by macroscopic measurement. Up to 6 mM of Ca2+ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba2+ closed the channel without affecting the single channel conductance. Ba2+ closed the channel from the cis side. In addition to the above channel, a small cation-selective channel of about 40 pS was found.     

Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle.

Open-channel ion permeation properties were investigated for Ca++-activated K+ (CaK) channels in solutions of K+ and its analogues T1+, Rb+, and NH4+. Single CaK channels were inserted into planar lipid bilayers composed of neutral phospholipids, and open-channel current-voltage (I-V) relations were measured in symmetrical and asymmetrical solutions of each of these individual ions. For all concentrations studied, the zero-voltage conductance falls in the sequence K+ greater than T1+ greater than NH4+ greater than Rb+. The shape of the I-V curve in symmetrical solutions of a single permeant ion is non-ohmic and is species-dependent. The I-V shape is sublinear for K+ and T1+ and superlinear for Rb+ and NH4+. As judged by reversal potentials under bi-ionic conditions with K+ on one side of the bilayer and the test cation on the other, the permeability sequence is T1+ greater than K+ greater than Rb+ greater than NH4+ at 300 mM, which differs from the conductance sequence. Symmetrical mixtures of K+ or NH4+ with Rb+ show a striking anomalous mole fraction behavior, i.e., a minimum in single-channel conductance when the composition of a two-ion mixture is varied at constant total ion concentration. This result is incompatible with present models that consider the CaK channel a single-ion pore. In total, the results show that the CaK channel finely discriminates among K+-like ions, exhibiting different energy profiles among these species, and that several such ions can reside simultaneously within the conduction pathway.     

Permeation of internal and external monovalent cations through the catfish cone photoreceptor cGMP-gated channel

The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations.     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.     

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.     

The cation specificity of the potassium and gramicidin channels of muscle fiber membrane

Conductance ratios (Gi/Gk) and permeability ratios (Pi/Pk) for monovalent cations in frog muscle fibres have been defined under constant current conditions using a double sucrose gap method. Selectivity determined from potassium channel conductance is: K+ greater than Rb+ greater than Cs+ greater than greater than NH4+ greater than Na+ greater than Li+. In gramicidin channels both the permeability and conductance sequences are identical: NH4+ greater than Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+. In isotonic K+-sulfate solution with one-sided addition of external [Tl+] (2.5 x 10(-3)-20 x 10(-3) M), differences in the conductance and permeability ratios for gramicidin channel were observed.     

Selectivity and gating of the type L potassium channel in mouse lymphocytes

Type l voltage-gated K+ channels in murine lymphocytes were studied under voltage clamp in cell-attached patches and in the whole-cell configuration. The kinetics of activation of whole-cell currents during depolarizing pulses could be fit by a single exponential after an initial delay. Deactivation upon repolarization of both macroscopic and microscopic currents was mono-exponential, except in Rb-Ringer or Cs-Ringer solution in which tail currents often displayed "hooks," wherein the current first increased or remained constant before decaying. In some cells type l currents were contaminated by a small component due to type n K+ channels, which deactivate approximately 10 times slower than type l channels. Both macroscopic and single channel currents could be dissected either kinetically or pharmacologically into these two K+ channel types. The ionic selectivity and conductance of type l channels were studied by varying the internal and external permeant ion. With 160 mM K+ in the cell, the relative permeability calculated from the reversal potential with the Goldman-Hodgkin-Katz equation was K+ (identical to 1.0) greater than Rb+ (0.76) greater than NH4+ = Cs+ (0.12) much greater than Na+ (less than 0.004). Measured 30 mV negative to the reversal potential, the relative conductance sequence was quite different: NH4+ (1.5) greater than K+ (identical to 1.0) greater than Rb+ (0.5) greater than Cs+ (0.06) much greater than Na+, Li+, TMA+ (unmeasurable). Single channel current rectification resembled that of the whole-cell instantaneous I-V relation. Anomalous mole-fraction dependence of the relative permeability PNH4/PK was observed in NH4(+)-K+ mixtures, indicating that the type l K+ channel is a multi-ion pore. Compared with other K+ channels, lymphocyte type l K+ channels are most similar to "g12" channels in myelinated nerve.     

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.     

Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel

Monovalent cation selectivity has been characterized for the 3',5'-cyclic guanosine monophosphate (cGMP)-activated channel in vertebrate photoreceptor outer segment plasma membranes without divalent cations. Macroscopic currents in excised, inside-out patches were activated with saturating concentrations of cGMP (200 microM). Using a bi-ionic protocol with symmetrical 120 mM ion concentrations across the membrane, alkali metal ions and certain organic cations were substituted for sodium on the cytoplasmic face. The relative permeabilities, determined from shifts in the reversal potential (Erev), were NH4 much greater than Na greater than guanidinium greater than K greater than Li greater than Rb greater than Cs (3.34: 1.0: 0.97: 0.93: 0.92: 0.74: 0.50, respectively). Erev's were also measured as a function of [Na], [NH4], and [Cs], and the slope of the relation was -59.8, -52.1, and -49.1 mV/decade, respectively. The slopes for NH4 and Cs differ significantly from the Nernst-Planck prediction of -58.2 mV/decade expected for a single ion channel. Relative permeabilities were also determined for the alkali metal series of ions with 20 mM ionic concentrations on both sides of the membrane. The permeability sequence at 20 mM was unchanged, but the relative permeability for NH4 and Cs deviated significantly from the measurements at 120 mM with 1.46 and 0.75 ratios, respectively. The dependence of Erev on absolute concentrations and the deviation from Nernst-Planck predictions are best explained by multi-ion occupancy of the cGMP-activated channel. Selectivity was also examined by comparing the conductance ratios as a function of potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

Ion transport mediated by the valinomycin analogue cyclo(L-Lac-L-Val-D- Pro-D-Val)3 in lipid bilayer membranes

Cyclo(L-Lac-L-Val-D-Pro-D-Val)3 (PV-Lac) a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes by forming a 1:1 ion-carrier complex. The selectively sequence for PV-Lac is identical to that of valinomycin; i.e., Rb+ greater than K+ greater than Cs+ greater than or equal to NH+4 greater than Na+ greater than Li+. The steady-state zero-voltage conductance, G(0), is a saturating function of KCl concentration. A similar behavior was found for Rb+, Cs+, and NH+4. However, the ion concentration at which G(0) reaches a plateau strongly depends on membrane composition. The current-voltage curves present saturating characteristics, except at low ion concentrations of Rb+, K+, or Cs+. The ion concentration at which the saturating characteristics appear depends on membrane composition. These and other results presented in this paper agree with a model that assumes complexation between carrier and ion at the membrane-water interface. Current relaxation after voltage-jump studies were also performed for PV-Lac. Both the time constant and the amplitude of the current after a voltage jump strongly depend on ion concentration and membrane composition. These results, together with the stationary conductance data, were used to evaluate the rate constants of the PV-Lac-mediated K+ transport. In glycerolmonooleate they are: association rate constant, 2 x 10(6) M-1 s-1; dissociation rate constant, 4 x 10(5) s-1; translocation rate constant for complex, 5 x 10(4) s-1; and the rate of translocation of the free carrier (ks), 55 s-1. ks is much smaller for PV-Lac than for valinomycin and thus limits the efficiency with which the carrier is able to translocate cations across the membrane.     

Temperature dependence of ion permeation at the endplate channel

The dependence of acetylcholine receptor mean single-channel conductance on temperature was studied at garter snake twitch-muscle endplates using fluctuation analysis. In normal saline under conditions where most of the endplate current was carried by Na+, the channel conductance increased continuously from near 0 degrees C to approximately 23 degrees C with a Q10 of 1.97 +/- 0.14 (mean +/- SD). When 50% of the bath Na+ was replaced by either Li+, Rb+, or Cs+, the Q10 did not change significantly; however, at any temperature the channel conductance was greatest in Cs-saline and decreased with the ion sequence Cs greater than Rb greater than Na greater than Li. The results were fit by an Eyring-type model consisting of one free-energy well on the extracellular side of a single energy barrier. Ion selectivity appeared to result from ion-specific differences in the well and not in the barrier of this model. With a constant barrier enthalpy for different ions, well free-energy depth was greatest for Cs+ and graded identical to the permeability sequence. The correlation between increased well depth (i.e., ion binding) and increased channel conductance can be accounted for by the Boltzmann distribution of thermal energy.     

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.     

A discrete type of potassium channel conductance in snail neurons

The results of further investigations on a single potential dependent K+ channel are described. It was shown that ionic selectivity of the channel for monovalent ions is too high: Li+, Na+, and Cs+ are practically impermeant ions. Permeability of the channel for Rb+ is approximately 10 times less, and for Tl+ it is 2 times more than permeability for K+. Besides, it was found that open K+ channel has 16 multiple conductance levels.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Structure-function study on a de novo synthetic hydrophobic ion channel.

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore.