External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.     

Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.     

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.     

The Functional Surface Charge Density of a Fast K Channel in the Myelinated Axon of Xenopus laevis

The action of Mg²⁺ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mm Mg²⁺), calculated from measurements of the instantaneous current at Na reversal potential after 50–100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to −0.45 e nm⁻². Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels. Received: 11 December 1997/Revised: 24 April 1998     

The modulatory site for the action of gadolinium on surface charges and channel gating.

The gadolinium (Gd3+)-induced shift of potential dependence and modulated gating of Na and K channels were analyzed. In a previous investigation, we explained the shift in terms of pure screening (no binding) of fixed surface charges and the modulation by binding to modulatory sites on the channels. In the present paper, we have extended this model by including effects on the charge density of Gd3+ binding to the modulatory sites. From fitting the extended model to experimental data, the charge density was estimated to be -0.6 e nm-2, and the Gd(3+)-induced charge change to be +0.15 e nm-2, and the maximal scaling factor to be 7.5 for both Na and K channels. Intrinsic KD values for binding to the K and Na channels were estimated to be 140 and 380 mM, respectively. Estimations of the extracellular charge density, from primary structures of cloned channels, were found to be in agreement with estimations based on the present model. The modulatory site was suggested to be located at the cluster of negatively charged residues between the fifth transmembrane segment (S5) and the pore-forming region for both Na and K channels. These suggestions imply several testable predictions about different K channels.     

A dipeptidyl aminopeptidase-like protein remodels gating charge dynamics in Kv4.2 channels

Dipeptidyl aminopeptidase-like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853-18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the channel's voltage-sensing domain. To test this hypothesis, we implemented a robust experimental system to measure Kv4.2 gating currents and study gating charge dynamics in the absence and presence of DPPX-S. The results demonstrated that coexpression of Kv4.2 and DPPX-S causes a -26 mV parallel shift in the gating charge-voltage (Q-V) relationship. This shift is associated with faster outward movements of the gating charge over a broad range of relevant membrane potentials and accelerated gating charge return upon repolarization. In sharp contrast, DPPX-S had no effect on gating charge movements of the Shaker B Kv channel. We propose that DPPX-S destabilizes resting and intermediate states in the voltage-dependent activation pathway, which promotes the outward gating charge movement. The remodeling of gating charge dynamics may involve specific protein-protein interactions of the DPPX-S's transmembrane segment with the voltage-sensing and pore domains of the Kv4.2 channel. This mechanism may determine the characteristic fast operation of neuronal Kv4 channels in the subthreshold range of membrane potentials.     

Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.     

Extracellular potassium inhibits Kv7.1 potassium channels by stabilizing an inactivated state

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1–50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC₅₀ of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2–5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.     

Disinactivation of N-type inactivation of voltage-gated K channels by an erbstatin analogue

In some A-type voltage-gated K channels, rapid inactivation is achieved through the binding of an N-terminal domain of the pore-forming alpha-subunit or an associated beta-subunit to a cytoplasmic acceptor located at or near the channel pore using the ball-and-chain machinery (1-5). This inactivation involving the N terminus is known as N-type inactivation. Here, we describe an erbstatin (Erb) analogue as a small molecule inhibitor of the N-type inactivation in channels of Kv1.4 and Kv1.1+Kvbeta1. We show that this inhibition of inactivation (designated as "disinactivation") is potent and selective for N-type inactivation in heterologous cells (Chinese hamster ovary and Xenopus oocytes) expressing these A-type channels. In Chinese hamster ovary cells, Erb increased the inactivation time constant of Kv1.4 from 86.5 +/- 9.5 to 150 +/- 10 ms (n = 6, p < 0.0 1). Similarly, Erb increased the inactivation time constant of Kv1.1+Kvbeta1 from 10 +/- 0.9 to 49.3 +/- 7 ms (n = 7, p < 0.01). The EC(50) for disinactivating Kv1.1+Kvbeta1 was 10.4 +/- 0.9 microm (n = 2-9). Erb had no effect upon another A-channel, Kv4.3, which does not utilize the ball-and-chain mechanism. The mechanism of Erb-induced disinactivation was also investigated. Neither cysteine oxidation nor tyrosine kinase inhibition was involved. The results demonstrate that Erb can be used as a base structure to identify potent, selective small molecule inhibitors of intracellular protein-protein interactions, and that these disinactivators may offer another therapeutic approach to the treatment of seizure disorders.     

The inhibitory effect of copper ions on lymphocyte Kv1.3 potassium channels.

We applied the whole-cell patch-clamp technique to study the inhibitory effect of copper ions (Cu) on the activity of Kv1.3 channels expressed in human lymphocytes. Application of Cu reversibly inhibited the currents to about 10% of the control value in a concentration-dependent manner with the half blocking concentration of 5.28+/-0.5 microM and the Hill's coefficient of 3.83+/-0.18. The inhibitory effect was saturated at 10 microM concentration. The inhibition was time-dependent and it was correlated in time with a significant slowing of the current activation rate. In contrast the voltage dependence of activation was not changed by Cu as well as the inactivation kinetics. The inhibitory effect of Cu was voltage-independent. It was also unaffected by changing the extracellular pH in the range from 6.4 to 8.4, raising the extracellular potassium concentration to 150 mM and by changing the holding potential from -90 to -60 mV. The inhibitory effect of Cu was not changed in the presence of an equivalent concentration of Zn. Altogether, obtained data suggest that Cu inhibits Kv1.3 channels by a different mechanism than Zn and that Cu and Zn act on different binding sites. The inhibitory effect of Cu was probably due to a specific binding of Cu on binding sites on the channels. Possible physiological significance of the Cu-induced inhibition of Kv1.3 channels is discussed.     

Effects of exercise training and hypercholesterolemia on adenosine activation of voltage-dependent K+ channels in coronary arterioles

Coronary arterioles from hypercholesterolemic swine display attenuated adenosine-mediated vasodilatation that is attributable to the elimination of voltage-dependent K(+) (Kv) channel stimulation. For the present study, we tested the hypotheses that exercise training would correct impaired adenosine-induced dilatation in coronary arterioles from hypercholesterolemic pigs through restoration of adenosine activation of Kv channels and that vasodilatation to the receptor-independent adenylyl cyclase activator, forskolin, would also be attenuated in arterioles from hypercholesterolemic pigs. Pigs were randomly assigned to a control (NC) or high-fat, high-cholesterol (HC) diet for 20 wk. Four weeks after the diet was initiated, pigs from both groups were assigned to exercise training (Ex; 5 days/wk for 16 wk) or sedentary (Sed) protocols, resulting in four groups of pigs: NC-Sed, NC-Ex, HC-Sed, and HC-Ex. Arterioles ( approximately 150 mum) from both HC-Sed and HC-Ex pigs displayed impaired adenosine-mediated dilatation that was attributable to the elimination of 4-aminopyridine (4-AP; 1 mM)-sensitive Kv channel activation compared with NC counterparts. Arteriolar smooth muscle whole cell Kv currents were significantly reduced in HC-Sed compared with NC-Sed, although HC-Ex and NC-Ex did not differ. Forskolin-mediated dilatation was attenuated by 4-AP (1 mM) and in a concentration-dependent manner by tetraethylammonium (TEA; 0.1-1 mM) in NC-Sed but not HC-Sed. Further, TEA-sensitive Kv currents were diminished in cells of HC-Sed compared with NC-Sed pigs. Quantitative RT-PCR revealed similar expression levels of Kv3.1 and 3.3 in arterioles of NC-Sed and HC-Sed swine with undetectable expression of Kv1.1, 3.2, and 3.4. Taken together, these results suggest that hypercholesterolemia-mediated attenuation of adenosine-induced vasodilatation in coronary arterioles is not corrected by exercise training and is likely attributable to an impairment in the pathway coupling adenylyl cyclase with a highly TEA-sensitive Kv channel isoform(s).     

Inactivation as a new regulatory mechanism for neuronal Kv7 channels

Voltage-gated K(+) channels of the Kv7 (KCNQ) family have important physiological functions in both excitable and nonexcitable tissue. The family encompasses five genes encoding the channel subunits Kv7.1-5. Kv7.1 is found in epithelial and cardiac tissue. Kv7.2-5 channels are predominantly neuronal channels and are important for controlling excitability. Kv7.1 channels have been considered the only Kv7 channels to undergo inactivation upon depolarization. However, here we demonstrate that inactivation is also an intrinsic property of Kv7.4 and Kv7.5 channels, which inactivate to a larger extent than Kv7.1 channels at all potentials. We demonstrate that at least 30% of these channels are inactivated at physiologically relevant potentials. The onset of inactivation is voltage dependent and occurs on the order of seconds. Both time- and voltage-dependent recovery from inactivation was investigated for Kv7.4 channels. A time constant of 1.47 +/- 0.21 s and a voltage constant of 54.9 +/- 3.4 mV were determined. It was further demonstrated that heteromeric Kv7.3/Kv7.4 channels had inactivation properties different from homomeric Kv7.4 channels. Finally, the Kv7 channel activator BMS-204352 was in contrast to retigabine found to abolish inactivation of Kv7.4. In conclusion, this work demonstrates that inactivation is a key regulatory mechanism of Kv7.4 and Kv7.5 channels.     

KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Electrostatic domino effect in the Shaker K channel turret

Voltage-gated K channels are regulated by extracellular divalent cations such as Mg(2+) and Sr(2+), either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl(2) shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at approximately -20 mV and Kv2.1 channel open at approximately +5 mV. The aim of this study was to identify critical residues for this Mg(2+)-induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg(2+)-shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425 > 419 > 451. Charging 425 and 419 not only affect the Mg(2+)-induced G(V) shift with 5-6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel's working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4-thus, an electrostatic domino effect.     

Surface charge and properties of cardiac ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels are present in a wide variety of tissues. The sensitivity of these channels to closure by cytosolic ATP (ATPi) varies significantly among different tissues and even within the same tissue. The purpose of this study was to test the hypothesis that negative surface charges modulate the sensitivity of the KATP channels to ATPi by influencing surface potential in the vicinity of the ATP- binding site(s) of the channel. Unitary currents through KATP channels were measured in inside-out membrane patches excised from rabbit ventricular myocytes using the patch-clamp technique. Agents known to be effective at screening negative surface charges were applied to the cytosolic surface of the patches, and their effects on ATP sensitivity were examined. These agents included Mg2+ (2-15 mM), Ba2+ (2-10 mM), and the polycations protamine (0.01-10 microM), poly-L-lysine (500 microM), and poly-L-arginine (0.5 microM). The divalent cations and the various polycations all dramatically reduced the concentration of ATPi required to half-maximally suppress current through KATP channels (Kd), from approximately 100 microM in the absence of these agents to 1.6-8 microM in their presence. The effects were dose dependent. Protamine also reduced the sensitivity of KATP channels to block by cytosolic ADP. The sensitivity of KATP channels to block by ATP was independent of membrane potential, suggesting that the ATP-binding site is not located within the transmembrane voltage field. The effects of the polycation poly-L-lysine on ATP sensitivity were also independent of membrane potential or the direction (inward or outward) of current through KATP channels. In addition to increasing ATP sensitivity, Mg2+, Ba2+, and the polycations all caused dose-dependent block of inward and outward currents through KATP channels over similar concentration ranges as their effects on ATP sensitivity. The block of inward current by polycations was not associated with reduction of single-channel conductance or evidence of fast open channel block. However, the polycations did cause a modest reduction in single-channel conductance of outward current. These results are consistent with the presence of negative surface charges that reduce the local ATP concentration at the ATP-binding site(s) on the channel, relative to the bulk cytosolic ATP concentration. Screening these negative surface charges with divalent cations or polycations decreases the local ATP gradient, resulting in a decrease in the apparent Kd for ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

K+ activation of kir3.1/kir3.4 and kv1.4 K+ channels is regulated by extracellular charges

K+ activates many inward rectifier and voltage-gated K+ channels. In each case, an increase in K+ current through the channel can occur despite a reduced driving force. We have investigated the molecular mechanism of K+ activation of the inward rectifier K+ channel, Kir3.1/Kir3.4, and the voltage-gated K+ channel, Kv1.4. In the Kir3.1/Kir3.4 channel, mutation of an extracellular arginine residue, R155, in the Kir3.4 subunit markedly reduced K+ activation of the channel. The same mutation also abolished Mg2+ block of the channel. Mutation of the equivalent residue in Kv1.4 (K532) abolished K+ activation as well as C-type inactivation of the Kv1.4 channel. Thus, whereas C-type inactivation is a collapse of the selectivity filter, K+ activation could be an opening of the selectivity filter. K+ activation of the Kv1.4 channel was enhanced by acidic pH. Mutation of an extracellular histidine residue, H508, that mediates the inhibitory effect of protons on Kv1.4 current, abolished both K+ activation and the enhancement of K+ activation at acidic pH. These results suggest that the extracellular positive charges in both the Kir3.1/Kir3.4 and the Kv1.4 channels act as "guards" and regulate access of K+ to the selectivity filter and, thus, the open probability of the selectivity filter. Furthermore, these data suggest that, at acidic pH, protonation of H508 inhibits current through the Kv1.4 channel by decreasing K+ access to the selectivity filter, thus favoring the collapse of the selectivity filter.     

Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits.

Dendrotoxin (DTX) homologues are powerful blockers of K+ channels that contain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)4(beta)4 stoichiometry. DTXk inhibits potently Kv1.1-containing channels only, whereas alphaDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXk with native K+ channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site-directed mutants in which amino acids were altered in the 310-helix, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [125I]DTXk binding to Kv1. 1-possessing channels in rat brain membranes and blockade of the Kv1. 1 current expressed in oocytes were quantified. Also, the levels of inhibition of [125I]alphaDTX binding to brain membranes by the DTXk mutants were used to measure their high- and low-affinity interactions, respectively, with neuronal Kv1.2-containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the alpha-helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled alphaDTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 310-helical N-terminal region of DTXk was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to approximately 1246-fold reduction in the inhibitory potency for [125I]DTXk binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximately 16-fold).