Voltage-dependent Block Research Articles

Gating Effects of a Single-Residue Substitution in the Pore of NMDA Receptors

NMDA receptors are glutamate-gated ion-channels with high Ca2+-permeability, voltage-dependent block by extracellular Mg2+, and slow gating kinetics. These intrinsic attributes are critical to coincidence detection and synaptic plasticity at excitatory synapses in brain. The GluN1/GluN2A isoform (N1/N2A) predominates in adult brain; it has strong voltage-dependent Mg2+ block and the fastest kinetics of all isoforms. In macroscopic measurements, Mg2+ block generates a region of steep negative slope in the current-voltage (I/V) relationship at membrane potentials negative to −50 mV. In single-channel current records, the binding of one Mg2+ ion precludes conduction by other cations and results in a discrete, resolvable gap. In the presence of strong metal chelators (EDTA) and for receptors that carry a single-residue substitution (N596G) in the N2A subunit (N1/N2AN+1G), the macroscopic I/V plot reverts to its normal linear shape and the Mg2+-dependent gap is absent from single-channel traces. To investigate whether the N+1G substitution in the pore impinges on the receptor's gating kinetics, we recorded single-channel current traces from cell-attached patches of HEK-cells containing only one N1/N2AN+1G receptor and compared these with those recorded from wild-type receptors under similar conditions. Measurements done in the absence of extracellular Mg2+ (1 mM EDTA in the recording pipette) revealed that the N+1G substitution caused a ∼2-fold decrease in activity (Po, 0.31 ± 0.04, n = 10 vs. 0.65 ± 0.04, n = 12 for wild-type, p < 0.001) due to ∼2-fold shorter openings and ∼3-fold longer closures. Our results indicate that a perturbation in the pore that was intended to render the channels less sensitive to block by extracellular divalent cations also has a significant effect on the gating of NMDA receptors.

PH-Dependent Gating Mechanism of Kir2.1 Inward Rectifier K+ Channel Independent of Polyamine and Magnesium Block

Kir2.1 inward rectifier K+ channel shows a strong inward rectification due to a voltage-dependent block of the channel pore by intracellular cations, such as polyamines and Mg2+. In this study, we conducted experiments using inside-out patch membranes and found that Kir2.1 channel exhibits an extremely slow, voltage-dependent gating that depends on the cytoplasmic pH in the acidic range. This gating seemed to be unrelated to the block induced by polyamines that remained trapped near the cytoplasmic pore or by Mg2+ or impurities of EDTA contaminated in the cytoplasmic solution. Acidification of the cytoplasmic solution did not markedly affect the polyamine block of the wild-type Kir2.1 channel, indicating that the acidic residues lining the Kir2.1 pore (e.g. D172, E224 and E299), whose negative charges are known to contribute to polyamine binding sites, were not neutralized at acidic pHs. Thus, these negative charges did not seem to confer the pH sensitivity of the gating. However, when Kir2.1 channels bearing a mutation at these residues were tested, neutralization of D172 in the transmembrane region abolished the pH-dependent gating. The findings suggest that the gating may be caused by a pore block by an unknown molecule, bearing a positive charge at acidic pHs.

Atypical Functional Properties of GluK3-Containing Kainate Receptors

The properties of synaptic receptors determine their mode of action at presynaptic and postsynaptic loci. Here, we investigated the atypical biophysical properties of GluK3-containing kainate receptors, which contribute to presynaptic facilitation at hippocampal mossy fiber synapses. We show, using fast glutamate applications on outside-out patches and kinetic modeling, that the low sensitivity of GluK3 receptors for glutamate is attributable to fast desensitization of partially bound receptors. Consequently, these receptors can only be activated by fast transients of high glutamate concentration. In addition, GluK3 receptors are very sensitive to voltage-dependent block by intracellular spermine that precludes activation of substantial currents at potentials positive to -50 mV. Two specific residues within the channel pore define this high-affinity site. Finally, GluK3 are calcium permeable in the same way as unedited GluK2 receptors. These receptors present unique properties among AMPA/kainate receptors that could reflect a specialized presynaptic function.

Phosphorylation alters the pharmacology of Ca2+‐activated Cl‐ channels in rabbit pulmonary arterial smooth muscle cells

Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-activated Cl(-) channel (Cl(Ca)) blocker niflumic acid (NFA) produces a paradoxical dual effect on I(Cl(Ca)), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I(Cl(Ca)) were modulated by phosphorylation. I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 microM). Dephosphorylation enhanced the ability of 100 microM NFA to inhibit I(Cl(Ca)). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, approximately 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I(Cl(Ca)) in the range of 0.1-250 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP. Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl(Ca) channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.

Early biophysics of the NMDA receptor channel

The voltage-dependent block of NMDA receptor channels by external Mg2+ (Mgo) was first reported in 1984 in two papers (Mayer et al. 1984; Nowak et al. 1984) that have had a great influence on our understanding of the functional role of NMDA receptors. However, the observations also offered a new way to explain the ‘gating’ of a voltage-dependent conductance, and our paper of 1988 published in The Journal of Physiology aimed primarily at a biophysical description of the Mgo block based on single channel records (Ascher & Nowak, 1988). To characterize the Mgo block we followed the approach used by Neher & Steinbach (1978) to study how QX222 transforms into ‘bursts’ the single openings of nicotinic ACh receptor channels. Assuming that Mgo enters the NMDA receptor channel, binds to a blocking site situated deep in the membrane and can only leave to the outside after unbinding, we evaluated the rates of Mg2+ binding and unbinding in various Mg2+ concentrations and at various potentials. We then deduced from the voltage dependence of these rates the depth of the blocking site in the membrane. This depth was evaluated by a coefficient ‘δ’ that could vary between 0 and 1. Our value of δ was close to 1, suggesting that the blocking site was actually very close to the inner limit of the membrane. This value was somewhat higher than the values obtained by analysis of the I–V relations of whole cell currents by Mayer & Westbrook (1985). We also characterized at the single channel level the Ca2+ permeability of the NMDA receptor channel. We measured the shifts of the reversal potential in different external Ca2+ concentrations and deduced the ratio of the permeabilities of Ca2+ and monovalent cations from the Goldman–Hodgkin–Katz voltage equation. Our results agreed with the values obtained by Mayer & Westbrook (1987) using I–V relations for whole cell current. We also observed that an increase in external Ca2+ reduced the single channel conductance, indicating that Ca2+ permeates the channel more slowly than monovalent cations. Our evaluation of the depth of the Mgo blocking site was soon put in doubt by the observation that the value of δ we deduced for Mgo block was not easily reconciled with the voltage dependence of the block by internal Mg2+ (Mgi) (Johnson & Ascher, 1990). The ‘crossing of the deltas’ paradox was solved by Jon Johnson and his collaborators, who showed that access of Mg2+ to the channel is prevented when permeant ions bind at the outer surface of the membrane. In the model of Antonov & Johnson (1999) the δ for Mgo is now equal to 0.5. We should also acknowledge that our single channel recordings made us miss the ‘slow Mgo unblock’ which was later described by Spruston et al. (1995) on whole cell current relaxations following voltage jumps, modelled by Vargas-Caballero & Robinson (2004) and by Kampa et al. (2004), and shown to be NR2 subunit dependent by Clarke & Johnson (2006). Recently the same authors (Clarke & Johnson, 2008) have shown that the slow block is the consequence of a voltage dependent gating which does not require Mgo. From 1991 onward, the cloning of the NMDA receptor subunits radically renewed the study of Mg2+ block and Ca2+ permeability. It rapidly led to the identification of the key amino acids involved in the binding of Mg2+ (reviewed by Dingledine et al. 1999), and it also revealed the heterogeneity of Mg block among NMDA receptors subtypes (not yet well understood). Concerning the Ca2+ permeability, the use of calcium indicators has allowed direct comparison of the Ca2+ influx and the total current and thus evaluation of the ‘fractional Ca2+ current’ (Pf). When appropriate corrections are made, the value of Pf agrees very well with the predictions of the GHK equation (Schneggenburger 1996). The molecular structures responsible for the Ca2+ permeability have been partially identified and comprise both a deep site, the N site of the NR1 subunit, and a superficial site at the entrance of the channel, the DRPEER motif, also specific to the NR1 subunit (Watanabe et al. 2002). Despite all these advances, one cannot yet say that either Mg2+ block or Ca2+ permeation are understood at the molecular level. We still lack a structural model of the NMDA receptor channel, but it may not be too far away.

Using Lidocaine and Benzocaine to Link Sodium Channel Molecular Conformations to State-Dependent Antiarrhythmic Drug Affinity

Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block. Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes. The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed. These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

Dihydrostreptomycin Goes through the Mechano-Electric Transduction Channel in Chick Cochlear Hair Cells

Objective: This study evaluated the ability of dihydrostreptomycin (DHSM) to go through the mechano-electric transduction (MET) channels in hair cells under physiological conditions. Materials and Methods: Tall hair cells were isolated from the chick basilar membrane (cochlea). Mechanical stimulation was applied by a glass rod attached to a piezoelectric bimorph, and MET currents were recorded with a whole-cell patch technique. The voltage-dependent block of DHSM to MET channel was estimated by calculating the relative conductances (the ratio of MET current in DHSM saline to DHSM-free saline) at various membrane potentials. Results and Conclusion: At membrane potentials between –100 and +50 mV, DHSM behaves as a voltage-dependent blocker according to a partial block model. At membrane potentials more negative than –100 mV, however, DHSM blocking decreased. This finding differed from the partial block model, but indicated that DHSM escaped through the channel pore into the cytoplasm by acting as a permeant channel blocker due to the large electrical driving force.

Ca2+‐permeable AMPA receptors in central neurons

Ca²⁺‐permeable AMPA receptors in central neurons

Oxadiazolylindazole Sodium Channel Modulators are Neuroprotective toward Hippocampal Neurones

We report the discovery of a new class of neuroprotective voltage-dependent sodium channel modulators exemplified by (5-(1-benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)methanamine 11 (CFM1178). The compounds were inhibitors of [(14)C]guanidinium ion flux in rat forebrain synaptosomes and displaced binding of the sodium channel ligand [(3)H]BW202W92. 11 and the corresponding N(2)-benzyl isomer, 38 (CFM6058), demonstrated neuroprotective activity in hippocampal slices comparable to sipatrigine. CYP450 enzyme inhibition observed with 11 was reduced with 38. In electrophysiological experiments on dissociated hippocampal neurons, these two compounds caused use- and voltage-dependent block of sodium currents. Sodium channel isoform profiling against Na(v)1.1-1.8 demonstrated that the standard sodium channel blocker lamotrigine had modest activity against Na(v)1.1, while sipatrigine was generally more potent and less selective. 11 and 38 showed potent activity against Na(v)1.6, pointing to pharmacological block of this isoform being consistent with the neuroprotective effect. 38 also showed use dependent block of Na(v)1.6 in HEK cells.

Dihydropyridine block of voltage-dependent K + currents in rat dorsal root ganglion neurons

The dihydropyridines nifedipine, nimodipine and Bay K 8644 are widely used as pharmacological tools to assess the contribution of L-type voltage-gated Ca 2+ channels to a variety of neuronal processes including synaptic transmission, excitability and second messenger signaling. These compounds are still used in neuronal preparations despite evidence from cardiac tissue and heterologous expression systems that they block several voltage-dependent K + (Kv) channels. Both because these compounds have been used to assess the relative contribution of L-type Ca 2+ channels to several different processes in dorsal root ganglion (DRG) neurons and because a relatively wide variety of Kv channels present in other neuronal populations is present in DRG neurons, we determined the extent to which dihydropyridines block Kv currents in these neurons. Standard whole cell patch clamp techniques were used to study acutely disassociated adult rat DRG neurons. All three dihydropyridines tested blocked Kv currents in DRG neurons; IC 50 values (concentration resulting in an inhibition that is 50% of maximum) for nifedipine and nimodipine-induced block of sustained Kv currents were 14.5 and 6.6 μM, respectively. The magnitude of sustained current block was 44±1.6%, 60±2%, and 56±2.9% with 10 μM nifedipine, nimodipine and Bay K 8644, respectively. Current block was occluded by neither 4-aminopyridine (5 mM) nor tetraethylammonium (135 mM). Dihydropyridine-induced block of Kv currents was not associated with a shift in the voltage-dependence of current activation or inactivation, the recovery from inactivation, or voltage dependent block. However, there was a small use-dependence to the dihydropyridine-induced block. Our results suggest that several types of Kv channels in DRG neurons are blocked by mechanisms distinct from those underlying block of Kv channels in cardiac myocytes. Importantly, our results suggest that if investigators wish to explore the contribution of L-type Ca 2+ channels to neuronal function, they should consider alternative strategies for the manipulation of these channels than the use of dihydropyridines.

NMDA Receptor Activation by Spontaneous Glutamatergic Neurotransmission

Under physiological conditions N-methyl-D-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg(2+). Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approximately -67 mV). In long-duration stable recordings, we averaged a large number of miniature excitatory postsynaptic currents (mEPSCs, >100) before or after application of dl-2 amino 5-phosphonovaleric acid, a specific blocker of NMDA receptors. The difference between the two mEPSC waveforms showed that the NMDA current component comprises approximately 20% of the charge transfer during an average mEPSC detected at rest. Importantly, the contribution of the NMDA component was markedly enhanced at membrane potentials expected for the depolarized up states (approximately -50 mV) that cortical neurons show during slow oscillations in vivo. In addition, partial block of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor component of the mEPSCs did not cause a significant reduction in the NMDA component, indicating that potential AMPA receptor-driven local depolarizations did not drive NMDA receptor activity at rest. Collectively these results indicate that NMDA receptors significantly contribute to signaling at rest in the absence of dendritic depolarizations or concomitant AMPA receptor activity.

Kinetic analysis of voltage‐dependent potentiation and block of the glycine α3 receptor by a neuroactive steroid analogue

We examined the actions of a carboxylated analogue of pregnanolone ((3alpha,5beta)-20-oxopregnane-3-carboxylic acid; 3alphaCOOH5betaP) on receptors composed of glycine receptor alpha3 subunits, expressed in Xenopus oocytes. This analogue both inhibits and potentiates this receptor; potentiation increases with more negative membrane potentials while block increases with less negative membrane potentials. We used a second analogue ((3alpha,5beta)-3-hydroxymethylpregnan-20-one; 3alphaCH(2)OH5betaP) to examine the mechanism for voltage-dependent potentiation. This analogue potentiates but does not block the glycine alpha3 receptor. Steady-state responses and current relaxations following voltage jumps support the idea that the voltage dependence of potentiation indirectly arises from a voltage dependence for channel activation by glycine, rather than an intrinsic voltage dependence for potentiation. Potentiation results from a slowing of the channel deactivation rate. In the absence of steroid, at a low [glycine] current relaxations after a voltage jump show two exponential components, with a weighted average time constant of approximately 425 ms (-50 mV, 22 degrees C). The rate for channel deactivation increases at more negative potentials (e-fold per 170 mV) whereas activation decreases (e-fold per 230 mV). The probability a channel is active at a high [glycine] is greater than 0.9. The addition of 10 microM 3alphaCH(2)OH5betaP decreases the deactivation rate by 6.3-fold (-50 mV). Voltage-dependent block by 3alphaCOOH5betaP is consistent with simple open-channel block, with voltage dependence reflecting interactions of the charge on the analogue with the electrical field. Block and unblock have equal but opposite dependence on membrane potential, and the charge on 3alphaCOOH5betaP senses approximately 70% of the membrane field at the blocking site. The apparent forward rate for block, however, is very slow (2 x 10(5) m(-1) s(-1)).

Electrostatic Interactions Between Polyamines And Charged Adducts In The Kir Inner Cavity

Physiological regulation of conductance in Kir channels is accomplished by voltage-dependent block by intracellular cations (Mg2+, polyamines). We have investigated the fine details of polyamine binding in Kir channels, and report unique and unexpected effects of cysteine modification in the inner cavity. Introduction of positive charges near the spermine binding site in Kir6.2[N160D] channels alters the kinetic and steady-state properties of spermine block, although specific effects depend dramatically on both the modified position, and the properties of the modifying agent. MTSEA or MTSET modification of L157C, one helical turn above residue 160, dramatically reduces spermine affinity, and accelerates spermine unbinding. However, effects of MTSEA vs. MTSET modification of L164C, one helical turn below residue 160, are significantly divergent. MTSEA modification again reduces spermine affinity, whereas MTSET has no effect on steady-state spermine block, and slows spermine block and unblock. This stark difference between MTSEA and MTSET is attributed to interactions of the carboxylate sidechain at residue 160 with the primary amine of the ethylamine adduct (MTSEA), which are lost with the quaternarized ethyl-trimethylamine adduct introduced by MTSET modification. Thus, MTSEA modification of L164C reduces spermine block indirectly, by neutralization of the nearby â€∼rectification controller’ residue, rather than a direct interaction with spermine. In contrast, the chemistry of MTSET precludes this interaction, leaving spermine affinity unaltered. Importantly, MTSET modification of L164C reduces affinity for extended spermine analogs, whereas incorporation at a shallower site (S212C) again slows dissociation of extended blockers, shedding light on the localization of the trailing ends of polyamine analogs in the pore. Collectively, the data demonstrate the subtle effects of charge modification in the inner cavity on polyamine-mediated inward rectification, and confirm stable spermine binding between the rectification controller and the selectivity filter.

Physical Determinants of Strong Voltage Dependence of K+ Channel Block

Many pharmacological agents, as well as some endogenous biological molecules, act by blocking ion channels in a strongly voltage-dependent manner. In retrospect, the first and most dramatic example of such action is the “anomalous” voltage dependence of inwardly rectifying K+ (Kir) conductance discovered by Bernard Katz six decades ago. He observed that, contrary to the classic voltage-gated K+ conductance, the Kir conductance tends to zero with membrane depolarization but increases with hyperpolarization. In intact cells and within physiological voltage ranges, certain Kir channels are as steeply voltage dependent as Kv channels yet, unlike Kv channels, they have no inherent voltage sensors: the observed voltage sensitivity instead reflects voltage-dependent block of their ion pore by intracellular cations such as the polyamine spermine. Our group has proposed that the high valence associated with block of strong rectifiers primarily reflects the movement, not of tetravalent spermine itself, but of five K+ ions displaced by spermine across the steep electric field in the narrow K+ selectivity filter. We will present experimental evidence for key requirements of this blocker-K+ displacement model, and will discuss the essential features that render a pore-blocking process strongly voltage dependent.

KirBac1.1: It's An Inward Rectifying Potassium Channel

The structures of KirBac1.1 and KirBac3.1 have been used extensively to generate in silico homology models of eukaryotic Kir channels and to explore ion permeation, gating and drug-channel interactions by computational approaches. Functional studies of KirBac1.1 have been limited to 86Rb+ flux assays, but we have now succeeded in measuring voltage-clamp currents of KirBac1.1 reconstituted in giant liposomes. Using the patch-clamp technique, we recapitulate the results of 86Rb+ flux experiments, showing that KirBac1.1 currents are potassium-selective, blocked by barium, and inhibited by PIP2. These findings suggest that KirBac1.1 channels are functionally similar to eukaryotic Kir channels. Like the weak inward rectifiers Kir1.1 and Kir6.2, introduction of a negative charge at the “rectification controller” residue in the inner cavity of KirBac1.1 (I138D) confers strong inward rectification (i.e. steep voltage-dependent block by spermine). Steady state single channel currents of KirBac1.1 show multiple subconductance levels and gating modes in 150 mM symmetrical K+. However, similar to eukaryotic Kir channels, single channel amplitudes exhibit mild intrinsic inward rectification, with a maximum conductance at ∼56 pS (-100 mV), and open probability is higher at positive potentials. Multiple conductance states are still present in single channel currents of other permeant ions such as Rb+ and Tl+. However, similar to many K+ channels, including KcsA, Rb+ and Tl+ single channel currents show increased mean open time and decreased conductance. We find that KirBac1.1(T142C), equivalent to the Kir6.2 high P(o) mutant L164C, also has a high open probability and is effectively blocked by Cd+. These electrophysiological results confirm that KirBac1.1 is a bona fide inward rectifying K+ channel and a tractable model for study of the molecular basis of inward rectification, permeation and gating in eukaryotic Kir channels.

Rotenone reduces Mg 2+-dependent block of NMDA currents in substantia nigra dopamine neurons

Rotenone is a pesticide that has been successfully used to produce a rodent model of Parkinson's disease. We reported previously that rotenone potently augmented N-methyl- d-aspartate (NMDA)-evoked currents in rat dopamine neurons via a tyrosine kinase-dependent mechanism. In this study, we investigated the effect of rotenone on the current–voltage relationship of NMDA-induced currents in substantia nigra zona compacta neurons recorded with whole-cell patch pipettes in slices of rat brain. In a physiologic concentration of extracellular Mg 2+ (1.2 mM), a 30 min perfusion with rotenone (100 nM) produced a marked increase in current evoked by bath application of NMDA (30 μM), especially when measured at relatively hyperpolarized currents. In the presence of rotenone, NMDA currents lost the characteristic region of negative-slope conductance that is normally produced by voltage-dependent block by Mg 2+. The voltage-dependent effect of rotenone on NMDA currents was mimicked by a low extracellular concentration of Mg 2+ (0.2 mM) and was antagonized by a high level of Mg 2+ (6.0 mM). Moreover, we report that the tyrosine kinase inhibitor genistein blocked the ability of rotenone to augment NMDA receptor currents. These results suggest that rotenone potentiates NMDA currents by a tyrosine kinase-dependent process that attenuates voltage-dependent Mg 2+ block of NMDA-gated channels. These results support the hypothesis that an excitotoxic mechanism might participate in rotenone-induced toxicity of midbrain dopamine neurons.

Regulation of Antiarrhythmic Drug Propafenone Effects on the C-type KV1.4 Potassium Channel by PHo and K+

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4ΔN channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC50) was 121 µM/L. With high, normal, and low extracellular potassium concentrations, the changes of IC50 value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 µM/L, alkalization to PH 8.0 reduced it to 58 µM/L. The results suggest that propafenone blocks the Kv1.4ΔN channel in the open state and give some hints for an intracellular site of action.

Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (IHERG) and the tail currents of HERG (Itail). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both IHERG and Itail were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either IHERG or Itail. The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 µg/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of Itail progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.

Molecular Determinants of Multiple Effects of Nickel on NMDA Receptor Channels

Nickel (Ni2+) is a toxic metal that affects the function of several neuronal ionic channels. Ni2+ inhibits N-methyl-D: -aspartate receptor (NR) channel in a voltage-dependent manner, but also causes enhancement of NR2B-containing channel activity and voltage-independent inhibition of those containing NR2A. The present work was aimed to find the sites of Ni2+ interaction on the NR2A and NR2B subunits by expressing wild-type and mutated NRs in either HEK293 cells or Xenopus laevis oocytes. The point mutation N616G in the pore region of the NR2B subunit completely removed the voltage-dependent block. In NR2 subunits deleted for their entire amino terminal domain (ATD) and expressed with wild-type NR1 subunit, voltage-independent inhibition of NR2A-containing channels was not modified, but the potentiation effect was abolished in NR2B-containing channels. In the latter channels, potentiation of the current was also removed by H127A, D101A, D104A point mutations and by the double mutation H127AD101A, all located in lobe I of ATD, and reduced by the point mutation T233A in lobe II, suggesting that the interaction site that causes potentiation shares common determinants with the Zn2+ and ifenprodil binding sites. In contrast, in NR2A-containing channels, we postulate the existence of an additional divalent cation binding site in the M3-M4 extracellular loop. In these channels, the point mutation H801A in the NR2A subunit caused an important reduction of the voltage-independent block, with a 7-time increase in IC(50). The block was also partially, but not as prominently, reduced by the double mutation H705AH709A in the same region of NR1. This additional binding site can be responsible of specific heavy metal interaction with NR channels.

Amphiphilic Blockers Punch through a Mutant CLC-0 Pore

Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl−, the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than −80 mV, a phenomenon similar to the blocker “punch-through” shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at −80 mV in symmetrical 140 mM Cl−, and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl− reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential. That the shift of the CPA-dissociation rate follows the Cl− gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.