Inactivation of the Potassium Conductance and Related Phenomena Caused by Quaternary Ammonium Ion Injection in Squid Axons

Several analogues of the tetraethylammonium (TEA⁺) ion were injected into the giant axon of the squid, and the resultant changes in time course and magnitude of the potassium current (I_K) were studied. For all the analogues used, three of the ethyl side chains of TEA⁺ were left unchanged, while the fourth chain was either lengthened or shortened. Increasing the length of this chain increased binding to the blocking site in the channel by a factor of roughly two for each added CH₂ group. The effect on the rate of entry into the blocking site was relatively slight. Thus the concentration for half-suppression of g_K decreased by about the same factor of two for each added CH₂. All the analogues caused anomalous or ingoing rectification. The longest chain analogue used, pentyltriethylammonium ion, caused rapid inactivation of g_K, and this inactivation had properties quite similar to g_Na inactivation. The anomalous rectification and the g_K inactivation caused by these compounds have the same basic mechanism.     

Ionic channels through the axon membrane (a review)

Ionic channels are discrete sites at which the passive movement of ions takes place during nervous excitation. Three types of channels are distinguished. 1. Leakage channels that are permanently open to various cations. 2. Na channels that open promptly on depolarization but slowly close again (inactivate) on sustained depolarization and that are predominantly permeable to Na⁺ ions. 3. K channels that on depolarization open after some delay but stay open and that are mainly passed by K⁺ ions. The selectivity sequence of the Na channels of the squid axon (or frog nerve) is as follows: Na⁺ ≈ Li⁺>(T1+)>NH⁺ ₄?K⁺> Rb⁺, Cs⁺; that of K channels is: (T1+)>K⁺>Rb⁺>NH⁺ ₄?Na⁺, Cs⁺, Na channels are selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX), K channels by tetraethylammonium ions (TEA). Either channel type is reversibly blocked when one drug molecule binds to one site per channel, the equilibrium dissociation constant of these reactions being about 3×10^?9 MTTX (or STX) and 4×10^?4 M TEA, respectively. Because of their specificity and high affinity, TTX and STX are used to “titrate” the Na channels whose density appears to be of the order of 100/Μm². The “gates” of the channels operate as a function of potential and time but independent of the permeating ion species. Drugs (e.g. veratridine) and enzymes (e.g. pronase, applied intraaxonally) cause profound changes in the gating function of the Na channels without influencing their selectivity. This points to separate structures for gating and ion discrimination. The latter is thought to be, in part, brought about by a “selectivity filter” of which detailed structural ideas exist. Recent experiments suggest that the gates of the Na channels are controlled by charged particles moving within the membrane under the influence of the electrical field.     

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.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Negative conductance caused by entry of sodium and cesium ions into the potassium channels of squid axons

Internal Cs⁺, Na⁺, Li⁺, and, to a lesser degree, Rb⁺ interfere with outward current through the K pores in voltage clamped squid axons. Addition of 100 mM NaF to the perfusion medium cuts outward current for large depolarizations about in half, and causes negative conductance over a range of membrane voltages. For example, suddenly reducing membrane potential from +100 to +60 mv increases the magnitude of the outward current. Internal Cs⁺ and, to a small extent, Li⁺, also cause negative conductance. Na⁺ ions permeate at least 17 times less well through the K pores than K⁺, and Cs⁺ does not permeate measurably. The results strongly suggest that K pores have a wide and not very selective inner mouth, which accepts K⁺, Na⁺, Li⁺, Cs⁺, tetraethylammonium ion (TEA⁺), and other ions. The diameter of the mouth must be at least 8 A, which is the diameter of a TEA⁺ ion. K⁺ ions in the mouths probably have full hydration shells. The remainder of the pore is postulated to be 2.6–3.0 A in diameter, large enough for K⁺ and Rb⁺ but too small for Cs⁺ and TEA⁺. We postulate that Na⁺ ions do not enter the narrower part of the pore because they are too small to fit well in the coordination cages provided by the pore as replacements for the water molecules surrounding an ion.     

Gating Properties of Inward-Rectifier Potassium Channels: Effects of Permeant Ions

Two inward-rectifier K⁺ channels, ROMK2 (Kir1.1b) and IRK1 (Kir2.1), were expressed in Xenopus oocytes and their gating properties were studied in cell-attached membrane patches. The gating properties depended strongly on the ion being conducted (K⁺, NH₄ ⁺, Rb⁺, or Tl⁺), suggesting tight coupling between permeation and gating. Mean open times were strongly dependent on the nature of the conducted ion. For ROMK2 the order from the longest to the shortest times was K⁺ > Rb⁺ > Tl⁺ > NH₄ ⁺. For IRK1 the sequence was K⁺ > NH₄ ⁺ > Tl⁺. In both cases the open times decreased monotonically as the membrane voltage was hyperpolarized. Both the absolute values and the voltage dependence of closed times were dependent on the conducted species. ROMK2 showed a single closed state whose mean lifetimes were biphasic functions of voltage. The maxima were at various voltages for different ions. IRK1 had at least two closed states whose lifetimes decreased monotonically with K⁺, increased monotonically with Tl⁺, and were relatively constant with NH₄ ⁺ as the conducted ion. We explain the ion-dependence of gating by assuming that the ions bind to a site within the permeation pathway, resulting in a stable, ion-dependent, closed state of the channel. The patterns of voltage-dependence of closed-state lifetimes, which are specific for different ions, can be explained by variations in the rate at which the bound ions leave the pore toward the inside or the outside of the cell. Received: 18 April 2001/Revised: 28 June 2001     

TETRAETHYLAMMONIUM BINDING TO THE OUTER MOUTH OF THE KcsA POTASSIUM CHANNEL: IMPLICATIONS FOR ION PERMEATION

ABSTRACT

Extracellular tetraethylammonium (TEA⁺) inhibits the current carried out by K⁺ ions in potassium channels. Structural models of wild-type (WT) and Y82C KcsA K⁺ channel/TEA⁺ complexes are here built using docking procedures, electrostatics calculations and molecular dynamics simulations. The calculations are based on the structure determined by Doyle et al.¹¹Our calculations suggest that in WT, the TEA⁺ cation turns binds at the outer mouth of the selectivity filter, stabilized by electrostatic and hydrophobic interactions with the four Tyr⁸² side chains. Replacement of Tyr⁸² with Cys causes a decrease of the affinity of the cation for the channel, consistently with the available site-directed mutagenesis data¹⁶. An MD simulation in which K⁺ replaces TEA⁺ provides evidence that TEA⁺ binding site can accommodate a potassium ion, in agreement with the high-resolution structure recently reported by Zhou et al.²⁰     

Intersubunit Coupling in the Pore of BK Channels

The structural basis underlying the gating of large conductance Ca²⁺-activated K⁺ (BK) channels remains elusive. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open state. The sensitivities of channels to calcium and voltage were modified by some mutations and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca²⁺ or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel closed. Additionally, single-channel recordings indicate that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance. These findings provide a basis for understanding the structure and gating of the BK channel pore.High conductance, “big” K⁺ (BK)⁴ channels encoded by the Slo1 gene are widely expressed in many tissues and respond to both membrane depolarization and submembrane Ca²⁺ concentration ([Ca²⁺]_i) (1–4). Because BK channels have apparently evolved from voltage-dependent K⁺ (K_V) channels, they share many features in common with K_V channels such as a similar K⁺ selectivity filter and conserved ion conduction pore (5). On the other hand, detailed studies indicate that there are significant differences in the permeation and gating properties of K_V and BK channels. First, BK channels have the largest single-channel conductance among K⁺ channels under similar recording conditions (6), partly because of two negatively charged rings at the entrance to the intracellular vestibule of BK channels that are absent from many K_V channels (7). Second, the mouth of the BK pore on the cytoplasmic side appears to be larger than other K⁺ channels because the on-rates for channel block by quaternary ammonium (QA) ions are limited only by diffusion (8), whereas the on-rates for QA block of other K⁺ channels are substantially slower. Additionally, it has been proposed that the pore-lining S6 segment of BK channels may not act as a permeation gate (8) as in Shaker K_V channels (9). Therefore, the BK channel may have an unusual pore, and understanding the structural basis of the above differences could provide important insight into the permeation and gating properties of K⁺ channels.Despite the differences between BK and K_V channels, it is likely that the opening and closing of their pores involve similar conformational changes. Flexing of the inner S6 helix at or near a conserved glycine residue is thought to open the pore, whereas straightening closes it (10, 11). The inner helix of BK channels is potentially more flexible than that of K_V channels because it contains a twin glycine motif (Gly-310–Gly-311). Differences in permeation property, pore geometry, and gating could potentially be accounted for by differences in S6 flexibility as well as the specific side-chain properties and interactions that occur between pore-lining residues. In this study we focused on Leu-312 and Phe-315 because we inferred from sequence analogy with KcsA channels (12) and our previous studies of BK channels (13, 14) that they are two of the five pore-lining residues (the others being Leu-309, Val-319, and Ile-323), and they are near the putative gating hinge. Our results indicate that substitutions of Leu-312 or Phe-315 have profound effects on BK channel gating, consistent with the notion that these residues interact between different subunits to hold the channel closed. The interaction of these residues may also be important for coordinating conformational changes in different subunits to ensure the efficient and cooperative long-range coupling between channel subunits, which is probably a general property of allosterically regulated proteins. Moreover, a surprising loss of Ca²⁺- and voltage sensitivity that accompanies mutations of Leu-312 or Phe-315 raises the possibility that these residues play a broader role in allosteric communication between sensors and gates.     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Sodium Conductance Activation without Inactivation in Pronase-perfused Axons

A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (I_Na) and potassium ion current (I_K) pass through the membrane in separate channels, or through a single set of channels which conduct first I_Na and then I_K. In support of the latter hypothesis it has been noted that the sodium conductance (g_Na) decline, called inactivation, proceeds with about the same time course as the potassium conductance (g_K) increase. This could mean that Na⁺ selective channels are being converted into K⁺ selective channels. The hypothesis is especially interesting because of the possibility that the carrier postulated in active transport is convertible from Na⁺ to K⁺ selectivity¹. An explicit statement of the single channel hypothesis and the means for disproving it were given by Mullins². Because a single channel could not simultaneously conduct I_Na and I_K, disproof requires that membrane conductance (g_m) be made somehow to exceed the maximum value of g_Na or g_K. We report here that inactivation of g_Na can be destroyed fairly selectively by the action from inside the axon of the unspecific proteolytic enzymes of pronase. In many cases g_m after pronase treatment is greater than maximum g_K before treatment, making untenable the single channel hypothesis.     

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.     

Modeling squid axon K+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the K⁺ channel of the squid giant axon, including the rising phase of the ON gating charge and the Cole-Moore effect, is provided. Upon accounting for a significant feature distinguishing K⁺, Na⁺ and Ca^2 + channels from channel-forming peptides modeled in our previous 2016 BBA paper, the nucleation-and-growth kinetic model developed therein is extended to simulate ON ionic and gating currents of the K⁺ channel of the squid giant axon at different depolarization potentials by the use of only two free parameters. K⁺ channel opening is considered to proceed by progressive aggregation of single subunits, while they are moving their gating charge outward under depolarizing conditions within their tetrameric structure; K⁺ channel closing proceeds in the opposite direction, by repolarization-induced disaggregation of subunits, accompanied by inward movement of their gating charge.     

Ion channel inhibitors block caspase activation by mechanisms other than restoring intracellular potassium concentration

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na⁺ and loss of K⁺ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA⁺) and 4-aminopyridine-sensitive K⁺ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K⁺] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K⁺]_i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K⁺ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K⁺]_i associated with apoptosis.     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

An overview of the potassium channel family

Potassium channels, tetrameric integral membrane proteins that form aqueous pores through which K⁺ can flow, are found in virtually all organisms; the genomes of humans, Drosophila, and Caenorhabditis elegans contain 30-100 K⁺ channel genes each. The structure of a bacterial K⁺ channel, sequence comparisons with other channels and electrophysiological measurements have enabled conclusions about the mechanism of gating and ion flow to be drawn for many other channels.     

Characterization of the Driving Force as a Modulator of Gating in Cardiac ATP-sensitive K+ Channels — Evidence for Specific Elementary Properties

Single cardiac ATP-sensitive K⁺ channels and, comparatively, two other members of the inwardly rectifying K⁺ channel family, cardiac K⁺ _(ir) and K⁺ _(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K⁺ gradient for open-state kinetics of these K⁺ channels. The conductive state of K⁺ _(ATP) channels was defined as a function of the electrochemical K⁺ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) τ_open proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K⁺ _(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of τ_open. Since gating continued to operate at E _K , i.e., in the absence of K⁺ permeation through the pore, K⁺ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb⁺ whose interference with an externally located binding site stabilizes the open state. In K⁺ _(ir) and K⁺ _(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K⁺ gradient. Thus, K⁺ _(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus. Received: 24 March 1997/Revised: 24 April 1998