Altered outward K+ currents in Drosophila larval neurons of memory mutants rutabaga and amnesiac

K⁺ currents in cultured Drosophila larval neurons have been classified into four categories according to their inactivation time constants, relative amplitude, and response to K⁺ channel blockers 4‐AP and triethylammonium. The percentage (65%) of neurons displaying K⁺ currents which were reduced to 30% in amplitude by 5 mM cyclic adenosine monophosphate (cAMP) analog 8‐bromo‐cAMP in both Drosophila memory mutants rutabaga (rut) and amnesiac (amn) was significantly larger than that (50%) in wild type. This initial characterization provides evidence for altered K⁺ currents in both rut and amn mutants. Arachidonic acid, a specifical inhibitor of Kv4 family (shal) K⁺ channels, was found to inhibit K⁺ currents in cultured Drosophila neurons, suggesting the presence of shal channels in these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 158–170, 1999     

Ecdysteroid control of ionic current development in Manduca sexta motoneurons

The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca²⁺ and K⁺ current densities. To determine whether 20-HE controls the changes in Ca²⁺ and K⁺ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca²⁺ and K⁺ currents were measured after 1–4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca²⁺ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca²⁺ current levels were larger in 20-HE–treated than in untreated cells. By contrast, 20-HE did not affect the Ca²⁺ current amplitudes of larval leg motoneurons. Whole-cell K⁺ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca²⁺ currents in leg motoneurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 211–223, 1998     

Effects of amyloid peptides on A‐type K+ currents of Drosophila larval cholinergic neurons

Accumulation of amyloid (Aβ) peptides has been suggested to be the primary event in Alzheimer's disease. In neurons, K⁺ channels regulate a number of processes, including setting the resting potential, keeping action potentials short, timing interspike intervals, synaptic plasticity, and cell death. In particular, A‐type K⁺ channels have been implicated in the onset of LTP in mammalian neurons, which is thought to underlie learning and memory. A number of studies have shown that Aβ peptides alter the properties of K⁺ currents in mammalian neurons. We set out to determine the effects of Aβ peptides on the neuronal A‐type K⁺ channels of Drosophila. Treatment of cells for 18 h with 1 μM Aβ1‐42 altered the kinetics of the A‐type K⁺ current, shifting steady‐state inactivation to more depolarized potentials and increasing the rate of recovery from inactivation. It also caused a decrease in neuronal viability. Thus it seems that alteration in the properties of the A‐type K⁺ current is a prelude to the amyloid‐induced death of neurons. This alteration in the properties of the A‐type K⁺ current may provide a basis for the early memory impairment that was observed prior to neurodegeneration in a recent study of a transgenic Drosophila melanogaster line over‐expressing the human Aβ1‐42 peptide. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Reduced cardiomyocyte Na+ current in the age-dependent murine Pgc-1β−/− model of ventricular arrhythmia

Peroxisome proliferator-activated receptor-γ coactivator-1 deficient (Pgc-1β^−/−) murine hearts model the increased, age-dependent, ventricular arrhythmic risks attributed to clinical conditions associated with mitochondrial energetic dysfunction. These were accompanied by compromised action potential (AP) upstroke rates and impaired conduction velocities potentially producing arrhythmic substrate. We tested a hypothesis implicating compromised Na⁺ current in these electrophysiological phenotypes by applying loose patch-clamp techniques in intact young and aged, wild-type (WT) and Pgc-1β^−/−, ventricular cardiomyocyte preparations for the first time. This allowed conservation of their in vivo extracellular and intracellular conditions. Depolarising steps elicited typical voltage-dependent activating and inactivating inward Na⁺ currents with peak amplitudes increasing or decreasing with their respective activating or preceding inactivating voltage steps. Two-way analysis of variance associated Pgc-1β^−/− genotype with independent reductions in maximum peak ventricular Na⁺ currents from −36.63 ± 2.14 (n = 20) and −35.43 ± 1.96 (n = 18; young and aged WT, respectively), to −29.06 ± 1.65 (n = 23) and −27.93 ± 1.63 (n = 20; young and aged Pgc-1β^−/−, respectively) pA/μm² (p < 0.0001), without independent effects of, or interactions with age. Voltages at half-maximal current V*, and steepness factors k in plots of voltage dependences of both Na⁺ current activation and inactivation, and time constants for its postrepolarisation recovery from inactivation, remained indistinguishable through all experimental groups. So were the activation and rectification properties of delayed outward (K⁺) currents, demonstrated from tail currents reflecting current recoveries from respective varying or constant voltage steps. These current–voltage properties directly implicate decreases specifically in maximum available Na⁺ current with unchanged voltage dependences and unaltered K⁺ current properties, in proarrhythmic reductions in AP conduction velocity in Pgc-1β^−/− ventricles.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Sodium-dependent plateau potentials in electrocytes of the electric fish <Emphasis Type="Italic">Gymnotus carapo</Emphasis>

The weakly electric fish Gymnotus carapo emits a triphasic electric organ discharge generated by muscle-derived electrocytes, which is modified by environmental and physiological factors. Two electrode current clamp recordings in an in vitro preparation showed that Gymnotus electrocytes fired repetitively and responded with plateau potentials when depolarized. This electrophysiological behavior has never been observed in electrocytes from related species. Two types of plateaus with different thresholds and amplitudes were evoked by depolarization when Na⁺-dependent currents were isolated in a K⁺- and Ca²⁺-free solution containing TEA and 4-AP. Two electrode voltage clamp recordings revealed a classical fast activating–inactivating Na⁺ current and two persistent Na⁺-dependent currents with voltage-dependencies consistent with the action potential (AP) and the two plateaus observed under current clamp, respectively. The three currents, the APs and the plateaus were reduced by TTX, and were absent in Na⁺-free solution. The different Na⁺-dependent currents in Gymnotus electrocytes may be targets for the modifications of the electric organ discharge mediated by environmental and physiological factors.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Dynamics of chloride and potassium currents during the action potential in Chara studied with action potential clamp

TheCl^– and K⁺ currents underlying the action potential (AP) in the giant alga Chara were directly recorded with the action potential clamp method. An electrically triggered action potential was recorded and repetitively replayed as command voltage to the same cell under voltage clamp. The resulting clamp current was close to zero. Only the initial rectangular current used for stimulation was approximately reproduced by the clamp circuit. Inhibition of Cl^– channels with niflumic acid or ethacrynic acid and of K⁺ channels with Ba²⁺ evoked characteristic compensation currents because the amplifier had to add the selectively inhibited currents. Integration of the compensation currents revealed a mean flux through Cl^– and K⁺ channels of 3.3 10^–6 and 2.1 10^–6 mole M^–2 AP^–1 respectively. The dynamics of CI^– and K⁺ channel activation/inactivation were obtained by converting the relevant clamp currents to ionic permeabilities using the Goldman-Hodgkin-Katz current equation. During the AP the Cl^– permeability reaches a peak 370 ms, on average, after termination of the stimulating pulse. The following inactivation proceeds 3.6 times slower than the activation. The increase in K⁺ permeability lags behind the rise in Cl^– permeability, reaching a peak approximately 2 s after the latter.     

Complex interactions among residues within pore region determine the K+ dependence of a KAT1‐type potassium channel AmKAT1

KAT1‐type channels mediate K⁺ influx into guard cells that enables stomatal opening. In this study, a KAT1‐type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1‐type channels, its activation is strongly dependent on external K⁺ concentration, so it can be used as a model to explore the mechanism for the K⁺‐dependent gating of KAT1‐type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5–pore–S6 region controls the K⁺ dependence of AmKAT1, and residue substitutions show that multiple residues within the S5–Pore linker and Pore are involved in its K⁺‐dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K⁺ dependence. Finally, we analyzed the potential mechanism for the K⁺ dependence of AmKAT1, which could originate from the requirement of K⁺ occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K⁺‐dependent gating of KAT1‐type channels.     

Multiple effects of protein kinase C activators on Na+ currents in mouse neuroblastoma cells

The effects of externally applied different protein kinase C (PKC) activators on Na⁺ currents in mouse neuroblastoma cells were studied using the perforated-patch (nystatin-based) whole cell voltage clamp technique. Two diacylglycerol-like compounds, OAG (1-oleoyl-2-acetyl-sn-glycerol), and DOG (1-2-dioctanoyl-rac-glycerol) attenuated Na⁺ currents without affecting the time course of activation or inactivation. The reduction in Na⁺ current amplitude caused by OAG or DOG was dependent on membrane potential, being more intense at positive voltages. The steady-state activation curve was also unaffected by these substances. However, both OAG and DOG shifted the steady-state inactivation curve of Na⁺ currents to more hyperpolarized voltages. Surprisingly, phorbol esters did not affect Na⁺ currents. Cis-unsaturated fatty acids (linoleic, linolenic, and arachidonic) attenuated Na⁺ currents without modifying the steady-state activation. As with DOG and OAG, cis-unsaturated fatty acids also shifted the steady-state inactivation curve to more negative voltages. Interestingly, inward currents were more effectively attenuated by cis-fatty acids than outward currents. Oleic acid, also a cis-unsaturated fatty acid, enhanced Na⁺ currents. This enhancement was not accompanied by changes in kinetic or steady-state properties of currents. Enhancement of Na⁺ currents caused by oleate was voltage dependent, being stronger at negative voltages. The inhibitory or stimulatory effects caused by all PKC activators on Na⁺ currents were completely prevented by pretreating cells with PKC inhibitors (calphostin C, H7, staurosporine or polymyxin B). By themselves, PKC inhibitors did not affect membrane currents. Trans-unsaturated or saturated fatty acids, which do not activate PKC's, did not modify Na⁺ currents. Taken together, the experimental results suggest that PKC activation modulates the behavior of Na⁺ channels by at least three distinct mechanisms. Because qualitatively different results were obtained with different PKC activators, it is not clear how Na⁺ currents would respond to activation of PKC under physiological conditions.This work was supported in part by a grant-in-aid from the American Heart Association (National Center), and by Loyola University Medical Center. Dr. Godoy is a recipient of a fellowship from Conselho Nacional de Pesquisas e Desenvolvimento (Brazil).     

Direct modulation of Na+ currents by protein kinase C activators in mouse neuroblastoma cells

We investigated the effects of different protein kinase C (PKC) activators on Na⁺ currents using the conventional whole-cell and the inside-out macropatch voltage-clamp techniques in mouse neuroblastoma cells (N1E-115). Two different categories of PKC activators were investigated: the cis-unsaturated fatty acids (CUFAs): oleic (cis-9-octadecenoic), linoleic (cis-9-12-octadecadienoic), and linolenic acid (cis-9-12-15-octadecatrienoic), and, the diacylglycerol (DAG) derivative 1-2-dioctanoyl-sn-glycerol (DOG). These substances caused the following alterations on Na⁺ currents: (i) Na⁺ currents were attenuated as a function of voltage. While DOG attenuated both inward and outward Na⁺ currents in a monotonic and continuous voltage-dependent manner, CUFAs preferentially attenuated inward currents; (ii) the steady-state activation curve of Na⁺ currents shifted to more depolarized voltages; (iii) opposite to the activation curve, the steady-state inactivation curve of Na⁺ channels (h curve) shifted to more hyperpolarized voltages; (iv) the time course of inactivation development was accelerated by PKC activators, while the recovery from inactivation was not affected; (v) substances that inhibit different metabolic pathways (PKC activation, cyclooxygenase, lipooxygenase, and P-450 pathways) did not prevent the effects of PKC activators on Na⁺ currents. One fully saturated fatty acid (octadecanoic acid), a trans-unsaturated fatty acid (trans-9-octadecenoic), and different phorbol esters did not affect Na⁺ currents; (vi) effects of different PKC activators on Na⁺ currents were completely reversible. These observations suggest that PKC activators might interact with Na⁺ channels directly. These direct effects must be taken into consideration in evaluating the overall effect of PKC activation on Na⁺ channels. Moreover, it is likely that this direct interaction could account, at least in part, for the diversity of effects of PKC activators on Na⁺ channels.This work was supported in part by a grant-in-aid from the American Heart Association (National Center).     

Properties of voltage-gated potassium currents of microglia differentiated with granulocyte/macrophage colony-stimulating factor

Voltage-gated whole-cell currents were recorded from cultured microglial cells which had been developed in the presence of the macrophage/microglial growth factor granulocyte/macrophage colony-stimulating factor. Outward K⁺ currents (I _K) were most prominent in these cells. I _Kcould be activated at potentials more positive than –40 mV. Half-maximal activation of I _Kwas achieved at –13.8 mV and half-maximal inactivation of I _Kwas determined at –33.8 mV. The recovery of I _Kfrom inactivation was described by a time constant of 7.9 sec. For a tenfold change in extracellular K⁺ concentration the reversal potential of I _Kshifted by 54 mV.Extracellularly applied 10 mm tetraethylammonium chloride reduced I _K by about 50%, while 5 mm 4-aminopyridine almost completely abolished I _K. Several divalent cations (Ba²⁺, Cd²⁺, Co²⁺, Zn²⁺) reduced current amplitudes and shifted the activation curve of I _Kto more positive values. Charybdotoxin (IC₅₀ = 1.14 nm) and noxiustoxin (IC₅₀=0.89 nm) blocked I _Kin a concentration-dependent manner, whereas dendrotoxin and mast cell degranulating peptide had no effect on the current amplitudes.     

K<Superscript>+</Superscript> Channels in Cardiomyocytes of the Pulmonate Snail <Emphasis Type="Italic">Helix</Emphasis>

We used the patch-clamp technique to identify and characterize the electrophysiological, biophysical, and pharmacological properties of K⁺ channels in enzymatically dissociated ventricular cells of the land pulmonate snail Helix. The family of outward K⁺ currents started to activate at –30 mV and the activation was faster at more depolarized potentials (time constants: at 0 mV 17.4 ± 1.2 ms vs. 2.5 ± 0.1 ms at + 60 mV). The current waveforms were similar to those of the A-type family of voltage-dependent K⁺ currents encoded by Kv4.2 in mammals. Inactivation of the current was relatively fast, i.e., 50.2 ± 1.8% of current was inactivated within 250 ms at + 40 mV. The recovery of K⁺ channels from inactivation was relatively slow with a mean time constant of 1.7 ± 0.2 s. Closer examination of steady-state inactivation kinetics revealed that the voltage dependency of inactivation was U-shaped, exhibiting less inactivation at more depolarized membrane potentials. On the basis of this phenomenon, we suggest that a channel encoded by Kv2.1 similar to that in mammals does exist in land pulmonates of the Helix genus. Outward currents were sensitive to 4-aminopyridine and tetraethylammonium chloride. The last compound was most effective, with an IC₅₀ of 336 ± 142 µmol l^–1. Thus, using distinct pharmacological and biophysical tools we identified different types of voltage-gated K⁺ channels. Present address for S.A.K.: Brigham and Womens Hospital, Cardiovascular Division, Harvard Medical School, 75 Francis St., Thorn 1216, Boston, MA 02115.     

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels : Evidence for Cation Binding Sites of Different Affinity

Voltage-gated potassium (K⁺) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K⁺ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K⁺ channel that is highly selective for K⁺ over Na⁺ in physiological solutions, but conducts Na⁺ in the absence of K⁺. Na⁺ and K⁺ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na⁺ currents inactivated more rapidly and deactivated more slowly than K⁺ currents. Currents carried by 160 mM Na⁺ were inhibited by external K⁺ with an apparent IC₅₀ <30 μM. K⁺ also altered both inactivation and deactivation kinetics of Na⁺ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K⁺ were inhibited by external Na⁺, with an apparent IC₅₀ of ∼100 mM. In contrast to the effects of low [K⁺] on Na⁺ current kinetics, Na⁺ did not affect K⁺ current kinetics, even at concentrations that inhibited K⁺ currents by 40–50%. These data suggest that Na⁺ block of K⁺ currents did not involve displacement of K⁺ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K⁺ channels, differential K⁺ and Na⁺ conductance, and the concentration dependence of K⁺ block of Na⁺ currents and Na⁺ block of K⁺ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.     

Dihydropyridine Action on Voltage-dependent Potassium Channels Expressed in Xenopus Oocytes

Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca²⁺ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K⁺ channels. We examined the effects of DHPs on the Shaker K⁺ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K⁺ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.