K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

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.     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Functional analysis of human Na~+/K~+-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes

AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine(FHM2, SHM). METHODS: cRNA of human Na+/K+-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the twoelectrode voltage-clamp(TEVC) technique on Xenopus oocytes. Stationary K+-induced Na+/K+ pump currents were measured, and the voltage dependence of apparent K+ affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na+ buffers to analyze kinetics and voltage-dependent presteady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant dif-ferences in the voltage dependence of K+-induced currents, and analysis of the transient currents indicated that the extracellular Na+ affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K+ interaction, D999H affected the voltage dependence of K+-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na+ was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.     

Kinetics of Na+, K+-ATPase Inhibition by a Rat Brain Endogenous Factor (II-E)

Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na⁺(1.56–200 mM), K⁺(1.25–40 mM), or ATP (1–8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter K_Mvalue, and suggested uncompetitive inhibition for Na⁺, K⁺or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.     

Mathematical modelling of ATP, K and Na interactions with (Na + K)-ATPase occurring under equilibrium conditions

The controlling effect of ATP, K⁺ and Na⁺ on the rate of (Na⁺ + K⁺)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.

1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.

2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K⁺ and Na⁺ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.

3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)₂ complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K⁺ and Na⁺ were absent or K⁺ and K⁺ plus Na⁺, respectively, were present in the experiments.

4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na⁺ + K⁺)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.

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).     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

Crystal structures of several bacterial Na_v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na_v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na_vAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Na_v channels. The non-equilibrium simulations indicate that two or three extracellular K⁺ ions can block the entrance to the selectivity filter of Na_vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K⁺ ions that can be ‘locked’ in place by modest applied forces. In contrast to K⁺, three Na⁺ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na⁺ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K⁺ block is equivalent to large applied potentials experimentally measured for two bacterial Na_v channels to induce inward currents of K⁺ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.     

Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

The Na⁺,K⁺-ATPase binds Na⁺ at three transport sites denoted I, II, and III, of which site III is Na⁺-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na⁺ affinity in the α1-, α2-, and α3-isoforms of Na⁺,K⁺-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na⁺-coordinating residues in site III. Remarkably, the Na⁺ affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na⁺ binding and does not affect the E₁-E₂ conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na⁺ affinity is likely intrinsic to the Na⁺ binding pocket, and the underlying mechanism could be a tightening of Na⁺ binding at Na⁺ site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na⁺,K⁺ pump function in intact cells. Rescue of Na⁺ affinity and Na⁺ and K⁺ transport by second-site mutation is unique in the history of Na⁺,K⁺-ATPase and points to new possibilities for treatment of neurological patients carrying Na⁺,K⁺-ATPase mutations.     

Interaction of local anesthetics with the K+ channel pore domain

Local anesthetics and related drugs block ionic currents of Na⁺, K⁺ and Ca²⁺ conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K⁺ channels, similar to the destabilization of channel function observed at low extracellular K⁺ concentration. Such drug-dependent stability may result from electrostatic repulsion of K⁺ from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K⁺ channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K⁺-dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K⁺. The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K⁺ channels.     

Human Adipose Cells Have Voltage-dependent Potassium Currents

The whole-cell patch-clamp method was used to study the membrane electrical properties of human adipocyte cells obtained by differentiating from precursors of human abdominal and mammary tissues. All differentiated cells exhibited outward currents with sigmoidal activation kinetics. The outward currents showed activation thresholds between –20 to –30 mV and slow inactivation. The ionic channels underlying the macroscopic current were highly selective for K⁺. Their selectivity was for typical K⁺ channels with relative permeabilities of K⁺>NH ₄ ⁺ >Cs⁺>Na⁺. No evidence of any other type of voltage-gated channel was found. The potassium currents (I _KV) were blocked reversibly by tetraethylammonium and barium. The IC ₅₀ value and Hill coefficient of tetraethylammonium inhibition of I _KV were 0.56 mM and 1.17 respectively. These results demonstrate that human adipose cells have voltage-dependent potassium currents.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.