Conduction properties of the cloned Shaker K+ channel.

The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously.     

Direct Measurement of the K+-Channel and Its Ion Selectivity in the Thylakoid Membrane from Spinach (Spinacea oleracea L.) Leaves

Thylakoid vesicles were purified from spinach (Spinacea oleracea L. ) leaves by sucrose density gradient centrifugation and incorporated into planar lipid bilayers by stirring. At least one type of voltage-dependent K+ single-channel currents was found. Its conductance (between +60 mV and –60 mV ) was about 55 pS in symmetrical (cis: trans) 250 mmol/L KC1. This channel was sensitive to TEA (tetraethylammonium chloride) and the permeability ratio (PK+/PCl-) was about 14. 9. The selectivity of 55 pS channel determined from both reversal potentials under bi-ionic conditions or from conductance measurements in symmetrical solutions, was in the seguence of K+〉Na+〉Li+ 〉NH4+〉 Cs+. This potassium channel could act as involved in charge-balancing during light-driven proton uptake by thylakoid.     

Single channel properties of P2X2 purinoceptors

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of approximately 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was approximately 150 mM at 0 mV and voltage dependent. The binding site appeared to be approximately 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 microM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of approximately 7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose-response curve.     

Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers.

The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule.     

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

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.     

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.     

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.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).     

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.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

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.     

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Use-dependent block of sodium channels in frog myelinated nerve by tetrodotoxin and saxitoxin at negative holding potentials

Na+ currents were measured in myelinated frog nerve fibres in the presence of nanomolar concentrations of tetrodotoxin (TTX) or saxitoxin (STX) in the extracellular solution. The Na+ currents declined during a train of depolarizing pulses if the fibre was held at hyperpolarizing potentials between the pulses. At a pulse frequency of 0.8 Hz, the peak Na+ currents were reduced to 70 or 60% of the initial value in 9.3 nM TTX and 3.5 nM STX solutions, respectively. A decline of Na+ currents was also observed in two-pulse experiments. The peak Na+ current during a second test pulse did not depend on the duration (0.2 to 12 ms) of the first pulse. It decreased with increasing interval between the pulses, reached a minimum and increased again. The results are interpreted with a use-dependent blockage of Na+ channels by TTX or STX at negative holding potentials. The effects were described quantitatively, assuming a fast affinity increase of toxin receptors at Na+ channels triggered by Na+ activation followed by slow toxin binding to channels and relaxation of the receptor affinity.     

Properties of the Ca-activated K conductance of human red cells as revealed by the patch-clamp technique

Application of Ca2+ to the inner surface of red-cell membranes activates unitary currents that can be measured in cell-attached and cell-free membrane patches. Ca2+ can be replaced by Pb2+ to activate the single channels. In addition to internal Ca2+ external K+ has to be present. The channels are preferentially permeable to K+ with a selectivity ratio PK:PNa of about 15:1 as estimated from measurement of reversal potentials. The dependence of channel activity on Ca2+ is compatible with the conception that the binding of two Ca2+ is necessary to open a single channel. Both the channel activity and the single-channel conductance exhibit inward rectification. External and internal Na+ inhibit the K+ currents. The reported results suggest that the unitary current events are responsible for the Ca2+-dependent K+ permeability known from measurement on cell suspensions. Therefore, comparison of the two techniques allows calculation of the number of K+ channels per red cell, which on average is about 10.