Open Channel Current Research Articles

Impact on AChR open channel noise by pore-peripheral salt bridge depends on voltage and divalent cations

Mechanisms behind the fluctuations in the ionic current through single acetylcholine receptor (AChR) channels have remained elusive. In a recent study of muscle AChR we showed that mutation of a conserved intramembrane salt bridge in the β- and δ-subunits markedly increased fluctuations in the open channel current that extended from low to high frequency. Here, we show that extracellular divalent cations reduce the high-frequency fluctuations and increase the low-frequency fluctuations. The low-frequency fluctuations are shown to arise from steps between two current levels, with the ratio of the time at each level changing e-fold for a 70 mV increase in membrane potential, indicating modulation by a charged element within the membrane field. Increasing the charge on the ion selectivity filter biases the ratio of current levels equivalent to a 50 mV increase in membrane potential but does not alter the voltage dependence of the ratio. The magnitudes of the voltage dependence and voltage bias allow estimates of the distance between the ion selectivity filter and the voltage-sensing element. Studies with either calcium or magnesium show that the two divalent cations synergize to increase the low-frequency fluctuations, whereas they act independently to decrease the high-frequency fluctuations, indicating multiple divalent cation binding sites. Molecular dynamics simulations applied to the structure of the Torpedo AChR reveal that mutation of the salt bridge alters the equilibrium positions and dynamics of residues local to the site of the mutation and within the adjacent ion selectivity filter in a calcium-dependent manner. Thus, disruption of a conserved intramembrane salt bridge in the muscle AChR induces fluctuations in open channel current that are sensitive to divalent cation binding at multiple sites and modulated by a charged element within the membrane field.

A novel AlGaN/GaN heterostructure field-effect transistor based on open-gate technology

In this study, a novel AlGaN/GaN heterostructure field-effect transistor based on open-gate technology was fabricated. Sample transistors of different structures and sizes were constructed. Through measurements, it was found that by changing the width of the opening, the threshold voltage of the device could be easily modulated across a larger range. The open-gate device had two working modes with different transconductance. When the gate-source voltage VGS ≤ − 4.5 V, only the open region was conductive, and a new working mechanism modulated the channel current. Corresponding theoretical analysis and calculations showed that its saturation mechanism was related to a virtual gate formed by electron injection onto the surface. Also, the gate-source voltage modulated the open channel current by changing the channel electron mobility through polarization Coulomb field scattering. When used as class-A voltage amplifiers, open-gate devices can achieve effective voltage amplification with very low power consumption.

Silver Nanoneedle Probes Enable Sustained DC Current, Single-Channel Resistive Pulse Nanopore Sensing.

Resistive pulse sensing using ion channel proteins (biological nanopores) has been evolving as a single-molecule approach to detect small biomolecules owing to atomically precise pore size reproducibility, high signal-to-noise ratio, and molecular selectivity. The incorporation of biological nanopores in sensing platforms requires a stable lipid membrane that can be formed by a variety of methods such as the painting method and droplet-based techniques. However, these methods are limited by the fragility of the unsupported bilayer or the need for specific microdevices. Electrode-supported bilayers, in which a metal electrode is used as a support structure, have been recently developed using a fine gold nanoneedle. We previously described the utility of the gold nanoneedle-supported ion channel probe to detect small molecules with high spatial resolution; however, it exhibited a channel current decay over time, which affected the binding frequency of the target molecule to the protein pore as well. Here, we introduce a silver nanoneedle probe to support the lipid bilayer formation and ion channel measurements. The silver nanoneedle mitigates the current decay observed on gold electrodes and produces stable DC channel currents. Our findings propose the formation of a AgCl layer creating a nonpolarizable electrode. The new nanoneedle is successfully applied for single-molecule detection of sulfonated β-cyclodextrin (S7βCD) using αHL as a test bed protein. We believe that this new silver nanoneedle platform has great potential given the relative ease of lipid bilayer formation and stable open channel currents.

Unmasking coupling between channel gating and ion permeation in the muscle nicotinic receptor.

Whether ion channel gating is independent of ion permeation has been an enduring, unresolved question. Here, applying single channel recording to the archetypal muscle nicotinic receptor, we unmask coupling between channel gating and ion permeation by structural perturbation of a conserved intramembrane salt bridge. A charge-neutralizing mutation suppresses channel gating, reduces unitary current amplitude, and increases fluctuations of the open channel current. Power spectra of the current fluctuations exhibit low- and high-frequency Lorentzian components, which increase in charge-neutralized mutant receptors. After aligning channel openings and closings at the time of transition, the average unitary current exhibits asymmetric relaxations just after channel opening and before channel closing. A theory in which structural motions contribute jointly to channel gating and ion conduction describes both the power spectrum and the current relaxations. Coupling manifests as a transient increase in the open channel current upon channel opening and a decrease upon channel closing.

Novel TRPA1 Antagonists are Multimodal Blockers of Human TRPA1 Channels: Drug Candidates for Treatment of Familial Episodic Pain Syndrome (FEPS)

The TRPA1 receptor‐channel is a therapeutic target for the discovery of novel pain drugs. In man, a single gain‐of‐function mutation in TRPA1, N855S, is known to be the cause of a familial episodic pain syndrome (FEPS). Multimodal activation by various stimuli is a fundamental feature of TRPA1 so that the precise manner in which TRPA1 and N855S‐TRPA1 is activated under pathophysiological conditions is not known, but is thought to be due to a combined effect of an array of inflammatory and nociceptive activators. Since the therapeutic potential of this class of drugs may depend on their efficacy to inhibit TRPA1 function by diverse activation mechanisms, we evaluated the activity of a group of novel TRPA1 antagonists, ALGX‐2542, 2567, 2582, 2586 and 2596 by multiple assay methods to determine mechanisms for inhibition and assess the therapeutic potential for this drug series. In the FLIPR‐calcium based pharmacological assays, the compounds demonstrated potent inhibition of either FFA or AITC induced activation of human TRPA1 (hTRPA1) expressed in HEK‐293 cells with IC50s ranging from 15–88 nM. The potent activity of these ALGX antagonists was confirmed in whole‐cell patch clamp electrophysiological assays. To demonstrate open channel block, hTRPA1 currents were first activated by AITC and upon reaching a pseudo‐steady state, each cell was exposed to a single concentration of an ALGX antagonist. All ALGX antagonists rapidly and completely blocked the open channel currents at 10‐fold their respective IC50 values. To compare open channel block and drug interactions with resting state channels, cells were pre‐incubated with ALGX antagonists for 5 min in the presence of external Ca2+, and then challenged with AITC. For this condition, all ALGX antagonists also completely blocked hTRPA1 activation, indicating that these compounds are effective inhibitors of both open and resting state hTRPA1 channels. Calcium imaging studies also showed that ALGX‐2542 potently inhibits activation by AITC, H202, and cold. The pharmacological activity of this series of antagonists was further defined for native human TRPA1 channels endogenously expressed in the WI‐38 human cell line. Utilizing the pre‐incubation FLIPR protocol, the series of ALGX antagonists was also found inhibit agonist‐induced increases in [Cai2+] with nearly equal IC50 values as found in the recombinant hTRPA1‐HEK assay system. The quantitative agreement (r2=0.95) demonstrates that the pharmacological activity defined in the recombinant assay systems directly translates to native hTRPA1 receptor‐channels. Furthermore, these antagonists also completely inhibited the AITC‐activation of the N855S hTRPA1 channels. Remarkably, the inhibitory effects of this series of antagonists is independent of the mode of TRPA1 activation, which includes structurally diverse agonists, H202, and cold temperatures with similar IC50s. This multimodal profile may be an optimal feature for producing maximal clinical efficacy for a pain drug targeting TRPA1. Our results identify a class of drugs that enable a therapeutic approach to treat pain episodes associated with FEPS.Support or Funding InformationThis work was supported by NIDA 1R44DA044900 to JMH and Algomedix.

Quantifying Short-Lived Events in Multi-State Ionic Channel Measurements

We present a new technique that considerably improves the resolution and accuracy of single molecule measurements with nanopores. Molecular interactions with nanopores are characterized by electrical measurements of discrete changes in the channel conductance. By representing physical components of the system with electrical equivalents, we used circuit theory to model response of the system to a stimulus (e.g., a molecule entering the channel). This allowed us to characterize short-lived events where the ionic current does not reach a steady state value, and were previously not analyzed. Applying this technique to measurements of poly(ethylene glycol) (PEG) molecules with the α-hemolysin (αHL) nanopore resulted in remarkable improvements in accuracy and the number of detected events. When measuring polydisperse PEG (mean molecular weights of 400 g/mol and 600 g/mol), the new method recovered ≈ 18-fold more events per unit time, compared with existing techniques, and discriminated molecules with as few as 8 monomers (PEG8). We validated the measurement of PEG with an αHL nanopore using results from a recently published study (Balijepalli et. al, J Am Chem Soc, 135: 7064, 2013) that refined a previous analytical theory with molecular dynamics simulations. Fitting this model to the newly obtained experimental data resulted in excellent agreement of both the blockade depth (the ratio of the ionic current when a molecule occupies the pore to the open channel current) and the residence times of the molecule in the channel, over the entire measurement range (PEG8 to PEG19). Finally, we applied the new analysis technique to recover the sequence of a known DNA strand with 26 bases, from a published ionic current trace (Manrao et. al, Nat. Biotechnol. 30: 349, 2012). The technique detected systematic fluctuations in the ionic current that were as small as 0.9 ± 0.04 pA.

50 Single-molecule studies of human telomeric G-quadruplexes and the effect of oxidative damage

In the human genome, telomeric DNA has tandem repeats of the sequence 5′-TTAGGG terminating with a 3′ single-stranded overhang of 100–200 bases. These guanine-rich DNA sequences can fold into tetrastranded structures, known as G-quadruplexes. The precise fold of the G-quadruplex structure is dictated by the metal ions present which we studied through the use of the α-hemolysin ion channel. Being electrophoretically driven into the cis side of the α-hemolysin, the hybrid fold (K+) entered the vestibule mouth leading to current blockages for the duration of the time the DNA resided in the vestibule. Due to the polymorphic nature of the hybrid folds, the recorded current signatures could be correlated with the major structural topologies that exist for this fold in solution (e.g. hybrid-1, hybrid-2, and triplex; Mashimo et al., 2010). The hybrid folds were not capable of traversing to the trans side of the nanopore, while the triplex could achieve translocation. The basket fold (Na+) was also able to enter into the vestibule causing current blockages that were indicative of the orientation in which they entered into the vestibule. When the basket fold entered tail first, slow translocation events were observed. In contrast, the propeller fold (∼3.9 nm, 0.05 M K+/5 M Li+) exceeds the protein channel orifice (∼3.0 nm) producing only swift and small disturbances to the open channel current. Secondly, oxidative damage to the telomeric sequence is proposed to contribute to telomere shortening, dysfunction, and cell aging (Epel et al., 2004). Locations of the oxidative damages have different effects on the G-quadruplex folding that produced significant changes in their nanopore behavior. Placement of the guanine oxidation product, 8-oxoguanosine (OG), in a top or bottom tetrad results in destabilization of that layer, whereas the presence of OG in a middle tetrad leads to complete unfolding of the G-quadruplex. These behaviors were determined by their translocation times which correlated with the folding’s free energy supported by NIH GM093099.

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave kon = 13 x 106 M−1s−1 and koff = 1609 sec−1 with KD = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a KD = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.

Single-Channel Current Through Nicotinic Receptor Produced by Closure of Binding Site C-Loop

We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed “resting” state, whereas the model after TMD simulation represents the channel in the proposed “active” state. Under external voltage biases, the channel in the “active” state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.

Functional Coupling between the Kv1.1 Channel and Aldoketoreductase Kvβ1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.

TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels.

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca(2+)-activated K(+) (K(Ca)) channels were present in inside-out patches excised from AH neurons. The P(o) of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca(2+)] from < 10 nM to either 500 nM or 10 microM. P(o) was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant K(Ca) channels measured approximately 60 pS under symmetric K(+) concentrations between -60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K(+)], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant K(Ca) channels by internal Ca(2+) in excised patches was not reversed by washing out the Ca(2+)-containing solution and replacing it with nominally Ca(2+)-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant K(Ca) channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca(2+) dependence of the activation of TEA- and apamin-resistant K(Ca) channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca(2+) to about 7 ms in the presence of submicromolar cytoplasmic Ca(2+). The Ca(2+)-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca(2+)-activated K(+) channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

Volatile anesthetics directly activate baseline S K + channels in Aplysia neurons

The actions of halothane on serotonin-sensitive potassium channels (S K + channels) were studied in sensory neurons of Aplysia. The normalized open probability of S K + channels was increased by clinical concentrations of halothane in cell-attached and excised patches from neurons of the pleural ventrocaudal cluster. No voltage-dependence of channel activation by halothane was observed. Pre-treatment of neurons with 8-bromo-cAMP (8-Br-cAMP) or nordihydroguaiaretic acid (NDGA) had no effect on the relative level of channel activation by halothane. S K + channels that were activated by arachidonic acid could also be activated by halothane and exhibited closely similar amplitude distributions of open channel current. Results from these experiments showed that S K + channel activation by halothane did not depend on second messenger modulation of channel activity. We conclude that it is likely that halothane directly activates S K + channels.

A novel slow hyperpolarization-activated potassium current (IK(SHA)) from a mouse hippocampal cell line.

1. A slow hyperpolarization-activated inwardly rectifying K+ current (IK(SHA)) with novel characteristics was identified from the mouse embryonic hippocampus x neuroblastoma cell line HN9.10e. 2. The non-inactivating current activated negative to a membrane potential of -80 mV with slow and complex activation kinetics (tau act approximately 1-7 s) and a characteristic delay of 1-10 s (-80 to -140 mV) that was linearly dependent on the membrane potential. 3. Tail currents and instantaneous open channel currents determined through fast voltage ramps reversed at the K+ equilibrium potential (EK) indicating that primarily K+, but not Na+, permeated the channels. 4. IK(SHA) was unaffected by altering the intracellular Ca2+ concentration between approximately 0 and 10 microM, but was susceptible to block by 5 mM extracellular Ca2+, Ba2+ (Ki = 0.42 mM), and Cs+ (Ki = 2.77 mM) 5. In cells stably transformed with M2 muscarinic receptors, IK(SHA) was rapidly, but reversibly, suppressed by application of micromolar concentrations of muscarine. 6. At the single channel level K(SHA) channel openings were observed with the characteristic delay upon membrane hyperpolarization. Analysis of unitary currents revealed an inwardly rectifying I-V profile and a channel slope conductance of 7 pS. Channel activity persisted in the inside-out configuration for many minutes. 7. It is concluded that IK(SHA) in HN9.10e cells represents a novel K+ current, which is activated upon membrane hyperpolarization. It is functionally different from both classic inwardly rectifying IKir currents and other cationic hyperpolarization-activated IH currents that have been previously described in neuronal or glial cells.

Kinetic analysis of Ca2+/K+ selectivity of an ion channel by single-binding-site models.

Current-voltage relationships of a cation channel in the tonoplast of Beta vulgaris, as recorded in solutions with different activities of Ca2+ and K+ (from Johannes & Sanders 1995, J. Membrane Biol. 146:211-224), have been reevaluated for Ca2+/K+ selectivity. Since conversion of reversal voltages to permeability ratios by constant field equations is expected to fail because different ions do not move independently through a channel, the data have been analyzed with kinetic channel models instead. Since recent structural information on K+ channels show one short and predominant constriction, selectivity models with only one binding site are assumed here to reflect this region kinetically. The rigid-pore model with a main binding site between two energy barriers (nine free parameters) had intrinsic problems to describe the observed current-saturation at large (negative) voltages. The alternative, dynamic-pore model uses a selectivity filter in which the binding site alternates its orientation (empty, or occupied by either Ca2+ or K+) between the cytoplasmic side and the luminal side within a fraction of the electrical distance and in a rate-limiting fashion. Fits with this model describe the data well. The fits yield about a 10% electrical distance of the selectivity filter, located about 5% more cytoplasmic than the electrical center. For K+ translocation, reorientation of the unoccupied binding site (with a preference of about 6:5 to face the lumenal side) is rate limiting. For Ca2+, the results show high affinity to the binding site and low translocation rates (<1% of the K+ translocation rate). With the fitted model Ca2+ entry through the open channel has been calculated for physiological conditions. The model predicts a unitary open channel current of about 100 fA which is insensitive to cytoplasmic Ca2+ concentrations (between 0.1 and 1 microM) and which shows little sensitivity to the voltage across the tonoplast.

Late sodium channel openings underlying epileptiform activity are preferentially diminished by the anticonvulsant phenytoin.

Late openings of sodium channels were observed in outside-out patch recordings from hippocampal neurons in culture. In previous studies of such neurons, a persistent sodium current appeared to underlie the ictal epileptiform activity. All the channel currents were blocked by tetrodotoxin. In addition to the transient openings of sodium channels making up the peak sodium current, there were two types of late channel openings: brief late and burst openings. These late channel openings occurred throughout voltage pulses that lasted 750 ms, producing a persistent sodium current. At -30 mV, this current was 0.4% of the peak current. The late channel openings occurred throughout the physiological range of trans-membrane voltages. The anticonvulsant phenytoin reduced the late channel openings more than the peak currents. The effect on the persistent current was greatest at more depolarized voltages, whereas the effect on peak currents was not substantially voltage dependent. In the presence of 60 microM phenytoin, peak sodium currents at -30 mV were 40-41% of control, as calculated using different methods of analysis. Late currents were 22-24% of control. Phenytoin primarily decreased the number of channel openings, with less effect on the duration of channel openings and no effect on open channel current. This set of findings is consistent with models in which phenytoin binds to the inactivated state of the channel. The preferential effect of phenytoin on the persistent sodium current suggests that an important pharmacological mechanism for a sodium channel anticonvulsant is to reduce late openings of sodium channels, rather than reducing all sodium channel openings. We hypothesize that pharmacological interventions that are most selective in reducing late openings of sodium channels, while leaving early channel openings relatively intact, will be those that produce an anticonvulsant effect while interfering minimally with normal function.

Study of neutron damage in GaAs MESFETs

Implanted channel GaAs MESFETs subjected to neutron irradiation show large modifications of the pinch-off voltage, open-channel saturation current, and transconductance in agreement with previous results. In this work we demonstrate how an experimental technique, based on the frequency dispersion of the transconductance, g/sub m/(f), and output conductance, g/sub D/(f), can identify the deep levels induced by neutron irradiation through measurements performed directly on packaged devices. After irradiation, a frequency dispersion of the transconductance has been observed, while it was flat in the unirradiated device. The g/sub m/(f) curve shape depends on the device bias conditions, and it has permitted for the first time to evaluate the activation energy of different deep levels induced by neutron irradiation in MESFETs.

Large-conductance calcium-activated potassium channels of cultured rat melanotrophs.

A large conductance, Ca(2+)-activated K+ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was approximately 190 pS and the probability (Po) of finding the channel in the open state at the resting membrane potential was low (< < 0.1). Channels in inside-out patches and in symmetrical 150 mM K+ had a conductance of approximately 260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K+ concentration of approximately 113 mM. The permeability sequence, relative to K+, was K+ > Rb+ (0.87) > NH4+ (0.17) > Cs+ > or = Na+ (< or = 0.02). The slope conductance for Rb+ was much less than for K+. Neither Na+ nor Cs+ carried measurable currents and 150 mM internal Cs+ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA+) produced a fast block for which the dissociation constant at 0 mV (KD(0 mV)) was 50 mM. The KD(0 mV) for external TEA+ was much lower, 0.25 mM, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA+ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nM) caused a large and reversible decrease of Po. The Po was increased by depolarization and/or by increasing the concentration of internal Ca2+. In 0.1 microM Ca2+ the half-maximal Po occurred at approximately 100 mV; increasing Ca2+ to 1 microM shifted the voltage for the half-maximal Po to -75 mV. The Ca2+ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca2+ binding sites on the BK channel.

Voltage‐dependent block by internal Ca2+ ions of inwardly rectifying K+ channels in guinea‐pig ventricular cells.

1. The block of the inwardly rectifying K+ channel by intracellular Ca2+ was studied in guinea-pig ventricular cells. 2. Single-channel currents through the inwardly rectifying K+ channel were recorded in the inside-out configuration at 150 mM external and internal K+. Internal Ca2+, at a concentration of 0.4-10 microM, induced subconductance levels with one-third and two-thirds of the unitary amplitude in the outward currents without affecting the inward currents. 3. Occupancy at each sublevel was estimated from the amplitude histogram which showed four equally spaced peaks in the presence of internal Ca2+. At different degrees of blockade, the distribution of the current levels showed a reasonable agreement with the binomial theorem. 4. The outward mean open-channel currents were measured at different Ca2+ concentrations and voltages. The current-voltage relation rectified inwardly in the presence of internal Ca2+ in a concentration-dependent manner. 5. The outward mean open-channel currents were normalized to unitary amplitudes in the absence of Ca2+. The normalized current-Ca2+ concentration curve was fitted by saturation kinetics with a Hill coefficient of 1 at each voltage. The voltage dependence of the dissociation constants gives the value for the fractional electrical distance of the Ca2+ binding site of 0.7. 6. The dwell times in each substrate were distributed exponentially. On the assumption that the inwardly rectifying K+ channel of cardiac cells is composed of three identical conducting subunits and each subunit is blocked by Ca2+ independently, the blocking (mu) and unblocking (lambda) rates were calculated. The value of mu increased with higher Ca2+ concentrations or larger depolarizations, while lambda was independent of Ca2+ and decreased with larger depolarization. 7. It is thus concluded that internal Ca2+ produces a voltage-dependent block of the channel to cause inward rectification although the blocking effect is less potent than that of Mg2+. The substate behaviour seen with internal Ca2+ supports the triple-barrelled structure of the cardiac inwardly rectifying K+ channel.

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition.

The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzymatic kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K+ over Rb+ and to its blockage by Cs+ and by Ca2+. The channel was investigated by patch-clamp techniques over a range of membrane voltages (Vm, referred to an extracytoplasmic electrical potential of zero) from -200 mV to +200 mV under various ionic conditions (0 to 300 mM K+, Rb+, Cs+, Ca2+, and Cl-) on the two sides of isolated patches. The experimental data are apparent steady-state current-voltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs+. The used model for K+ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K(+)-loaded and charged, facing the cytoplasm, (2) K(+)-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. Vm enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10(6)s-1 for zero voltage and 1 M substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 15,800. For the additional presence of an alternate transportee (here Rb+), the model can be extended in an analog way by another two states ((5) Rb(+)-loaded and charged, facing cytoplasm, and (6) Rb(+)-loaded and charged, facing vacuole) and six more rate constants (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs+ and Ca2+). k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 M-1 x exp(Vm/(64 mV)), kvac(Ca2+): 10 M-1 x exp(-Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 M-2 x exp(-Vm/(12.25 mV)).(ABSTRACT TRUNCATED AT 400 WORDS)

Fast and slow blockades of the inward-rectifier K+ channel by external divalent cations in guinea-pig cardiac myocytes

The present patch-clamp study shows that external Mg2+, Ca2+ and Sr2+ decrease the unit amplitude of inward current through the inward-rectifier K+ channel in a concentration-dependent manner. Sr2+ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr2+ binding site (delta) is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg2+ or Ca2+ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg2+ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs+ block (delta = 1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs+ and Sr2+ bind.