Tl+-induced micros gating of current indicates instability of the MaxiK selectivity filter as caused by ion/pore interaction

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.     

Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+

Inward rectifier (IR) currents were studied in bovine pulmonary artery endothelial cells in the whole-cell configuration of the patch-clamp technique with extracellular K+ concentrations, [K+]o, ranging from 4.5 to 160 mM. Whether the concentration of free Mg2+ in the intracellular solution, [Mg2+]i, was 1.9 mM or nominally 0, the IR exhibited voltage- and time-dependent gating. The IR conductance was activated by hyperpolarization and deactivated by depolarization. Small steady-state outward IR currents were present up to approximately 40 mV more positive than the K+ reversal potential, EK, regardless of [Mg2+]i. Modeled as a first-order C in equilibrium O gating process, both the opening rate, alpha, and the closing rate, beta, were exponentially dependent on voltage, with beta more steeply voltage dependent, changing e-fold for 9 mV compared with 18 mV for an e-fold change in alpha. Over all [K+]o studied, the voltage dependence of alpha and beta shifted along with EK, as is characteristic of IR channels in other cells. The steady-state voltage dependence of the gating process was well described by a Boltzmann function. The half-activation potential was on average approximately 7 mV negative to the observed reversal potential in all [K+]o regardless of [Mg2+]i. The activation curve was somewhat steeper when Mg-free pipette solutions were used (slope factor, 4.3 mV) than when pipettes contained 1.9 mM Mg2+ (5.2 mV). The simplest interpretation of these data is that IR channels in bovine pulmonary artery endothelial cells have an intrinsic gating mechanism that is not due to Mg block.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

Pore structure influences gating properties of the T-type Ca2+ channel alpha1G

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I-IV of the alpha1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the alpha1G subunit (CaV3.1) to those of pore mutants containing aspartate-to-glutamate substitution in domains III (EEED) or IV (EEDE). The change of the extracellular pH induced similar effects on the activation properties of alpha1G and both pore mutants, indicating that the larger affinity of the mutant channels for protons is not the cause of the gating modifications. Both mutants showed alterations in several gating properties with respect to alpha1G, i.e., faster macroscopic inactivation in the voltage range from -10 to 50 mV, positive voltage shift and decrease in the voltage sensitivity of the time constants of activation and deactivation, decrease of the voltage sensitivity of the steady-state inactivation, and faster recovery from inactivation for long repolarization periods. Kinetic modeling suggests that aspartate-to-glutamate mutations in the EEDD locus of alpha1G modify the movement of the gating charges and alter the rate of several gating transitions. These changes are independent of the alterations of the selectivity properties and channel protonation.     

Slowing of sodium channel opening kinetics in squid axon by extracellular zinc

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.     

Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.     

Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels.

We studied the effect of monovalent thallium ion (Tl(+)) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (tau = 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl(+) must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current. These results suggest that Tl(+) ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K(+) with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl(+) greatly diminishes Ba(2+) block. The unblocking rate of Ba(2+) is increased by >22-fold when the external permeant ion is switched from K(+) to Tl(+) regardless of the direction of Ba(2+) exit. This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.     

Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

Voltage-dependent K(+) channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K(+) channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca(2+) or Ba(2+), suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K(+)] (47 mV per 10-fold increase in [K(+)]), suggesting that K(+) binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K(+) ≈ Rb(+) > Cs(+) > Na(+) > Li(+) ≈ NMG(+). Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K(+)] using kinetic schemes in which the open-conductive state is stabilized by K(+) binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K(+) dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K(+)-sensitive inactivation gating, a property that may be common to other K(+) channels.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter

Using the lanthanide gadolinium (Gd(3+)) as a Ca(2+) replacing probe, we investigated the voltage dependence of pore blockage of Ca(V)1.2 channels. Gd(+3) reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd(3+) at concentrations used (<1 microM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose-response curves for the two blocking effects of Gd(3+) shifted in parallel for Ba(2+), Sr(2+), and Ca(2+) currents through the wild-type channel, and for Ca(2+) currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd(3+). The correlation indicates that Gd(3+) binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range -60 to -45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (-100 to -45 mV) and of use-dependent block (-40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd(3+) decreases with concentration of Ba(2+), indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd(3+) binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.     

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.     

The anomalous mole fraction effect in Chara: gating at the edge of temporal resolution

The anomalous mole fraction effect (AMFE) of the K(+) channel in excised patches of the tonoplast of Chara showed a minimum of apparent open-channel current at 20 mM Tl(+) and 230 mM K(+). Time series obtained at a sampling rate of 100 kHz (filter 25 kHz) were analyzed by three methods to find out whether the AMFE results from an effect on gating or on the conductivity of the open state. Fitting the amplitude histograms by a superposition of gaussians showed a broadening in the presence of Tl(+). Dwell-time analysis based on an O-O-C-C-C model failed to evaluate rate constants above the filter frequency. Thus, the absence of any reduction of apparent open-channel current in time series simulated with the evaluated rate constants could not be taken as evidence against the hypothesis of gating. Finally, a direct fit of the measured time series using five different 5-state Hidden Markov models revealed that the presence of Tl(+) changed the rate constants in such a way that the number of transitions into the short-lived open state (30 micros) increased strongly compared to those in the absence of Tl(+). These models explain 25% reduction of apparent single-channel current amplitude through a rapid gating mechanism.     

Modification of sodium channel gating by lanthanum. Some effects that cannot be explained by surface charge theory

In clonal pituitary (GH3) cells we studied the changes in sodium channel gating caused by substitution of La3+ for Ca2+ ion. Gating of sodium channels was simplified by using intracellular papain to remove inactivation. To quantify La effects, we empirically fitted closing and the late phase of opening of the channels with single exponentials, determined the opening (a) and closing (b) rate, and plotted these rates as a function of Vm (membrane voltage). The midpoint of the fraction open-Vm curve was also determined. Changing from Ca to La shifted the curves for these three measures of Na channel gating along the voltage axis and changed their shape somewhat. Surface charge theory, in the form usually presented, predicts equal shifts of all three curves, with no change in shape. We found, however, that the shift for each of the measurements was different. 2 mM La, for example, shifted opening kinetics by +52 mV (i.e., 52 mV must be added to the depolarization to make activation in 2 mM La as fast as in 2 mM Ca), the fraction open voltage curve by +42.5 mV, and the closing rate curve by +28 mV. The shift was an almost linear function of log [La] for each of the measures. The main finding is that changing from 2 mM Ca to 10 microM La causes a positive shift of the opening rate and fraction open curves, but a negative shift of the closing rate curve. The opposite signs of the two effects cannot be explained in terms of surface charge theory. We briefly discuss some alternatives to this theory.     

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.     

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.     

Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part II: Neutralization of aspartate 292 reduces long channel openings and gating current slow component

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.     

CFTR displays voltage dependence and two gating modes during stimulation

The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP- dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at - 100 mV, while the slow Lorentzian was voltage independent. In multi- channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.