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Molecular simulation techniques were appplied to predict the interaction of the voltage-dependent Shaker potassium channel with the channel-blocking toxin kappa-conotoxin-PVIIA (PVIIA). A structural thee-dimensional model of the extracellular vestibule of the potassium channel was constructed based on structural homologies with the bacterial potassium channel Kcsa, whose structure has been solved by X-ray crystallography. The docking of the PVIIA molecule was obtained by a geometric recognition algorithm, yielding 100 possible conformations. A series of residue-residue distance restraints, predicted from mutation-cycle experiments, were used to select a small set of a plausible channel-toxin complex models among the resulting possible conformations. The four final conformations, with similar characteristics, can explain most of the single-point mutation experiments done with this system. The models of the Shaker-PVIIA interaction predict two clusters of amino acids, critical for the binding of the toxin to the channel. The first cluster is the amino acids R2, I3, Q6 and K7 that form the plug of the toxin that interacts with the entrance to the selectivity filter of the channel. The second cluster of residues, R22, F23, N24 and K25, interacts with a channel region near to the external entrance of the pore vestibule. The consistency of the obtained models and the experimental data indicate that the Shaker-PVIIA complex model is reasonable and can be used in further biological studies such as the rational design of blocking agents of potassium channels and the mutagenesis of both toxins and potassium channels.

We describe the identification and characterization of a new gene deleted in the AMME contiguous gene syndrome. This gene is predominantly expressed in heart, skeletal muscle, spinal cord, and brain. Screening of placenta and NT2 cDNA libraries enabled us to obtain the 1.5-kb full-length transcript, which shows a 426-bp open reading frame. Since the resulting 142-amino-acid peptide has a single putative transmembrane domain and a weak but suggestive homology with KCNE1 (minK), a protein associated with the KCNQ1 potassium channel (KVLQT1), we named this new gene KCNE1-like (KCNE1L). To obtain greater insight into this new member of an apparently distinct protein family, we have identified and characterized the homologous mouse gene (Kcne1l), which encodes a peptide of 143 amino acids with 91% homology and 80% identity. The expression pattern of mouse Kcne1l in the developing embryo revealed strong signal in ganglia, in the migrating neural crest cells of cranial nerves, in the somites, and in the myoepicardial layer of the heart. The specific distribution in adult tissues, the putative channel function, and the expression pp6tern in the developing mouse embryo suggest that KCNE1L could be involved in the development of the cardiac abnormalities as well as of some neurological signs observed in patients with AMME contiguous gene syndrome., (Copyright 1999 Academic Press.)

We studied the phenomenon of cumulative inactivation in the voltage-dependent K+ channels of the Shaker-related subfamily Kv1 cloned from rat brain and expressed in Xenopus oocytes. In Kv1.4, repetitive stimulations at intervals shorter than 20 s produce cumulative inactivation even for brief stimuli that elicit K+ currents which do not show any significant decline during the depolarising pulse. These effects are absent or greatly reduced in the clones Kv1.1, Kv1.3, Kv1.5 and Kv1.6, and in the deletion mutant Kv1.4-delta-110, characterised by lack of "fast" (N-type) inactivation. We find that the inactivation caused by a single pulse increases after the pulse while the channels deactivate, and subsides with two time constants, indicating the existence of (at least) two inactivated states: IS, with a slow recovery kinetics and IF, with faster kinetics. In the simplest kinetic scheme accounting for our observations, IF is coupled sequentially to the open state O, while IS can be reached at a fast rate both from IF and from a pre-open, activated state, A, that is in fast equilibrium with O. The accumulation of long-lasting inactivation during the repolarisation is favoured by the prolongation of the lifetime of activated states due to the presence of IF. This explains the smaller accumulation effect observed in channels lacking fast inactivation. The physiological implications of these findings suggest how different channels of the Kv1 subfamily can affect differently the firing behaviour of neurones.

The modulation by protein kinase C (PKC) of the RCK1 K+ channel was investigated in Xenopus oocytes by integration of two-electrode voltage clamp, site-directed mutagenesis and SDS-PAGE analysis techniques. Upon application of beta-phorbol 12-myristate 13-acetate (PMA) the current was inhibited by 50-90%. No changes in the voltage sensitivity of the channel, changes in membrane surface area or selective elimination of RCK1 protein from the plasma membrane could be detected. The inhibition was mimicked by 1-oleoyl-2-acetyl-rac-glycerol (OAG) but not by alphaPMA, and was blocked by staurosporine and calphostin C. Upon deletion of most of the N-terminus a preceding enhancement of about 40% of the current was prominent in response to PKC activation. Its physiological significance is discussed. The N-terminus deletion eliminated 50% of the inhibition. However, phosphorylation of none of the ten classical PKC phosphorylation sites on the channel molecule could account, by itself or in combination with others, for the inhibition. Thus, our results show that PKC activation can modulate the channel conductance in a bimodal fashion. The N-terminus is involved in the inhibition, however, not via its direct phosphorylation.

We studied the temperature dependence of the currents mediated by potassium channels Kv1.1 cloned from rat brain expressed by permanent transfection in the human embryonic kidney cell line 292er. Voltage-gated outward currents, with a reversal potential near to the Nernst potential for potassium, were elicited by depolarising pulses. The currents activate fast and do not show inactivation during 50-ms test pulses. Increasing temperature decreased the amplitude of the currents, slowed the kinetics of activation and deactivation and shifted the activation curve to more negative potentials. We conclude that in Kv1.1 channels the closed states are associated with more ordered structure of the channel protein than the open state. The voltage-dependence and the kinetic properties are similar to those expressed in frog oocytes injected with cRNA coding the same Kv1.1.

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K+ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than -100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K+ channels and the activity of the plasma membrane H(+)-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H+ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg(2+)-free, 100 mM-K+, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium.

Activation of Cl- and K+ conductances by nucleotide receptor-operated mobilization of intracellular Ca2+ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltage-clamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl- ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1 +/- 0.3 min. Its amplitude was about 2.7 nA at -100 mV with 100 mumol/l of either ATP or UTP. The second current was carried by K+ ions and was blocked by Cs+. This current peaked more slowly and had a mean duration of 4.6 +/- 0.7 min. Its amplitude was 0.9 nA and 0.5 nA at -20 mV with 100 mumol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca2+ concentration ([Ca2+]i) that was similar in the presence or absence of extracellular Ca2+. The ED50 for UTP was 24 mumol/l and that for ATP was 94 mumol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by thapsigargin prevented both the nucleotide-induced [Ca2+]i increase and the activation of membrane currents. Addition of 2 mmol/l Ca2+ to thapsigargin-treated cells produced a sustained increase of Cl- and K+ currents, which was reversed by Ca2+ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca2+]i that stimulates Ca(2+)-dependent Cl- and K+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the activation properties of members of the Shaker-related subfamily of voltage-gated K+ channels cloned from rat brain and expressed in Xenopus oocytes. We find that Kv1.1, Kv1.4, Kv1.5, and Kv1.6 have similar activation and deactivation kinetics. The k+ currents produced by step depolarisations increase with a sigmoidal time course that can be described by a delay and by the derivative of the current at the inflection point. The delay tends to zero and the logarithmic derivative seems to approach a finite value at large positive voltages, but these asymptotic values are not yet reached at +80 mV. Deactivation of the currents upon stepping to negative membrane potentials below -60 mV is fairly well described by a single exponential. The decrease of the deactivation time constant at increasingly negative voltages tends to become less steep, indicating that this parameter also has a finite limiting value, which is not yet reached, however, at -160 mV. The various clones studied have very similar voltage dependencies of activation with half-activation voltages ranging between -50 and -11 mV and maximum steepness yielding and e-fold change for voltage increments between 3.8 and 7.0 mV. The shallower activation curve of Kv1.4 is likely to be due to coupling with the fast inactivation process present in this clone.

Macroscopic potassium currents were studied in cell-attached and inside-out patches from rat cerebellar granule cells. They were related with transient IA type potassium channels. Currents activated rapidly at potentials higher than -40 mV and did not inactivate completely. The magnitude of the current diminished when the membrane patches were excised. No differences in the activation and inactivation properties were found between patches in the integral cells and cell free membrane patches. A biophysical description of the currents is presented.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.

Brain fast transient K+ channel (A channel) is known to be modulated by PKC activation. We studied, by two-electrode voltage clamp, the molecular mechanism of modulation by PKC activation of A-channels expressed in Xenopus oocytes from the Shaker H4 clone. The modulation is inhibitory affecting primarily the maximal conductance of the channels. A secondary effect is a small change in the voltage-dependence of activation and inactivation of the channel.