Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

The ClC-3 Cl?/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl⁻/H⁺ antiport current (I_ClC-3) in HEK293 cells. The outward rectification and time dependence of I_ClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl⁻ current (ICl_acid) seen at extracellular pH ≤ 5.5. ICl_acid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl_acid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E_rev) of I_ClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl⁻ and H⁺ transport become uncoupled at low pH. At pH 4.0 E_rev was completely Cl⁻ dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native ICl_acid; 1) RNA interference directed at ClC-3 message reduced native ICl_acid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type I_ClC-3 and ICl_acid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I_ClC-3 and from native ICl_acid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl_acid. Low pH uncouples Cl⁻/H⁺ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl⁻/H⁺ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.     

Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174- 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163-174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603-626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316-2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance-voltage curve ( approximately 5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specific residue in the S4-S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404, 406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.     

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.     

The Inhibitory Effects of Ca2+ Channel Blocker Nifedipine on Rat Kv2.1 Potassium Channels

It is well documented that nifedipine, a commonly used dihydropyridine Ca²⁺ channel blocker, has also significant interactions with voltage-gated K⁺ (Kv) channels. But to date, little is known whether nifedipine exerted an action on Kv2.1 channels, a member of the Shab subfamily with slow inactivation. In the present study, we explored the effects of nifedipine on rat Kv2.1 channels expressed in HEK293 cells. Data from whole-cell recording showed that nifedipine substantially reduced Kv2.1 currents with the IC₅₀ value of 37.5 ± 5.7 μM and delayed the time course of activation without effects on the activation curve. Moreover, this drug also significantly shortened the duration of inactivation and deactivation of Kv2.1 currents in a voltage-dependent manner. Interestingly, the half-maximum inactivation potential (V _1/2) of Kv2.1 currents was -11.4 ± 0.9 mV in control and became -38.5 ± 0.4 mV after application of 50 μM nifedipine. The large hyperpolarizing shift (27 mV) of the inactivation curve has not been reported previously and may result in more inactivation for outward delayed rectifier K⁺ currents mediated by Kv2.1 channels at repolarization phases. The Y380R mutant significantly increased the binding affinity of nifedipine to Kv2.1 channels, suggesting an interaction of nifedipine with the outer mouth region of this channel. The data present here will be helpful to understand the diverse effects exerted by nifedipine on various Kv channels.     

Patch Clamp on the Luminal Membrane of Exocrine Gland Acini from Frog Skin (Rana esculenta) Reveals the Presence of Cystic Fibrosis Transmembrane Conductance Regulator–like Cl? Channels Activated by Cyclic AMP

Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a conductance of 10.0 ± 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 ± 6.7 mV), indicating that Cl⁻ was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out patches with 25 mM Cl⁻ on the inside, activity of small (8-pS) linear Cl⁻-selective channels was dependent upon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as P_Br > P_I > P_Cl from the Goldman equation; however, the conductance sequence when either halide was permeating the channel was G_Cl > G_Br >> G_I. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 ± 72 ms) and two closed states (lifetimes: 12 ± 4 and 224 ± 31 ms, respectively). Power density spectra had a double-Lorentzian form with corner frequencies 0.85 ± 0.11 and 27.9 ± 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regulator Cl⁻ channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267: C491–C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.     

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.     

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.     

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.     

A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions

Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Ménez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406-37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies DeltaDeltaGint obtained from double-mutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.     

Evidence for presence and functional effects of Kv1.1 channels in β-cells: general survey and results from mceph/mceph mice

Background

Voltage-dependent K⁺ channels (Kv) mediate repolarisation of β-cell action potentials, and thereby abrogate insulin secretion. The role of the Kv1.1 K⁺ channel in this process is however unclear. We tested for presence of Kv1.1 in different species and tested for a functional role of Kv1.1 by assessing pancreatic islet function in BALB/cByJ (wild-type) and megencephaly (mceph/mceph) mice, the latter having a deletion in the Kv1.1 gene.

Methodology/Principal Findings

Kv1.1 expression was detected in islets from wild-type mice, SD rats and humans, and expression of truncated Kv1.1 was detected in mceph/mceph islets. Full-length Kv1.1 protein was present in islets from wild-type mice, but, as expected, not in those from mceph/mceph mice. Kv1.1 expression was localized to the β-cell population and also to α- and δ-cells, with evidence of over-expression of truncated Kv1.1 in mceph/mceph islets. Blood glucose, insulin content, and islet morphology were normal in mceph/mceph mice, but glucose-induced insulin release from batch-incubated islets was (moderately) higher than that from wild-type islets. Reciprocal blocking of Kv1.1 by dendrotoxin-K increased insulin secretion from wild-type but not mceph/mceph islets. Glucose-induced action potential duration, as well as firing frequency, was increased in mceph/mceph mouse β-cells. This duration effect on action potential in β-cells from mceph/mceph mice was mimicked by dendrotoxin-K in β-cells from wild-type mice. Observations concerning the effects of both the mceph mutation, and of dendrotoxin-K, on glucose-induced insulin release were confirmed in pancreatic islets from Kv1.1 null mice.

Conclusion/Significance

Kv1.1 channels are expressed in the β-cells of several species, and these channels can influence glucose-stimulated insulin release.     

Disruption of Kv1.1 N-type inactivation by novel small molecule inhibitors (disinactivators)

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.     

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.     

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis

Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t_½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.     

A subfamily of acidic alpha-K(+) toxins

Three homologous acidic peptides have been isolated from the venom of three different Parabuthus scorpion species, P. transvaalicus, P. villosus, and P. granulatus. Analysis of the primary sequences reveals that they structurally belong to subfamily 11 of short chain alpha-K(+)-blocking peptides (Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) Trends Pharmacol. Sci. 20, 444-447). These toxins are 36-37 amino acids in length and have six aligned cysteine residues, but they differ substantially from the other alpha-K(+) toxins because of the absence of the critical Lys(27) and their total overall negative charge. Parabutoxin 1 (PBTx1), which has been expressed by recombinant methods, has been submitted to functional characterization. Despite the lack of the Lys(27), this toxin blocks several Kv1-type channels heterologously expressed in Xenopus oocytes but with low affinities (micromolar range). Because a relationship between the biological activity and the acidic residue substitutions may exist, we set out to elucidate the relative impact of the acidic character of the toxin and the lack of the critical Lys(27) on the weak activity of PBTx1 toward Kv1 channels. To achieve this, a specific mutant named rPBTx1 T24F/V26K was made recombinantly and fully characterized on Kv1-type channels heterologously expressed in Xenopus oocytes. Analysis of rPBTx1 T24F/V26K displaying an affinity toward Kv1.2 and Kv1.3 channels in the nanomolar range shows the importance of the functional dyad above the acidic character of this toxin.     

Characterization of a novel radiolabeled peptide selective for a subpopulation of voltage-gated potassium channels in mammalian brain.

BgK, a 37-amino acid voltage-gated potassium (Kv) 1 channel blocker isolated from the sea anemone Bunodosoma granulifera, can be modified at certain positions to alter its pharmacological profile (Alessandri-Haber, N., Lecoq, A., Gasparini, S., Grangier-Macmath, G., Jacquet, G., Harvey, A. L., de Medeiros, C., Rowan, E. G., Gola, M., Ménez, A., and Crest, M. (1999) J. Biol. Chem. 274, 35653-35661). In the present study, we report the design of two BgK analogs that have been radiolabeled with (125)INa. Whereas BgK(W5Y/Y26F) and its radiolabeled derivative, (125)I-BgK(W5Y/Y26F), bind to Kv1.1, Kv1.2, and Kv1.6 channels with potencies similar to those for the parent peptide, BgK, BgK(W5Y/F6A/Y26F) and its monoiodo-tyrosine derivative, (125)I-BgK(W5Y/F6A/Y26F), display a distinctive and unique pharmacological profile; they bind with high affinity to homomultimeric Kv1.1 and Kv1.6 channels, but not to Kv1.2 channels. Interaction of BgK(W5Y/F6A/Y26F) with potassium channels depends on the nature of a residue in the mouth of the channel, at a position that determines channel sensitivity to external tetraethylammonium. In native brain tissue, (125)I-BgK(W5Y/F6A/Y26F) binds to a population of Kv1 channels that appear to consist of at least two sensitive (Kv1.1 and/or Kv1.6) subunits, in adjacent position. Given its unique pharmacological properties, (125)I-BgK(W5Y/F6A/Y26F) represents a new tool for studying subpopulations of Kv1 channels in native tissues.     

Temporal changes of soil physic‐chemical properties at different soil depths during larch afforestation by multivariate analysis of covariance

Soil physic-chemical properties differ at different depths; however, differences in afforestation-induced temporal changes at different soil depths are seldom reported. By examining 19 parameters, the temporal changes and their interactions with soil depth in a large chronosequence dataset (159 plots; 636 profiles; 2544 samples) of larch plantations were checked by multivariate analysis of covariance (MANCOVA). No linear temporal changes were found in 9 parameters (N, K, N:P, available forms of N, P, K and ratios of N: available N, P: available P and K: available K), while marked linear changes were found in the rest 10 parameters. Four of them showed divergent temporal changes between surface and deep soils. At surface soils, changing rates were 262.1 g·kg⁻¹·year⁻¹ for SOM, 438.9 mg·g⁻¹·year⁻¹ for C:P, 5.3 mg·g⁻¹·year⁻¹ for C:K, and −3.23 mg·cm⁻³·year⁻¹ for bulk density, while contrary tendencies were found in deeper soils. These divergences resulted in much moderated or no changes in the overall 80-cm soil profile. The other six parameters showed significant temporal changes for overall 0–80-cm soil profile (P: −4.10 mg·kg⁻¹·year⁻¹; pH: −0.0061 unit·year⁻¹; C:N: 167.1 mg·g⁻¹·year⁻¹; K:P: 371.5 mg·g⁻¹ year⁻¹; N:K: −0.242 mg·g⁻¹·year⁻¹; EC: 0.169 μS·cm⁻¹·year⁻¹), but without significant differences at different soil depths (P > 0.05). Our findings highlight the importance of deep soils in studying physic-chemical changes of soil properties, and the temporal changes occurred in both surface and deep soils should be fully considered for forest management and soil nutrient balance.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b

The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex₃HexNAc₄Fuc₁ glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man₈GlcNAc₂ glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.     

Engineering-specific pharmacological binding sites for peptidyl inhibitors of potassium channels into KcsA

The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 [Legros, C., Pollmann, V., Knaus, H. G., Farrell, A. M., Darbon, H., Bougis, P. E., Martin-Eauclaire, M. F., and Pongs, O. (2000) J. Biol. Chem. 275, 16918-16924]. This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors. A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ([(125)I]KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ([(125)I]HgTX(1)(A19Y/Y37F)). The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels. The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments. KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides. KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques. Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels. This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.