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A bicyclic thioether analogue of alpha-conotoxin G1, a neurotoxin found in the venom of cone snails, was synthesized on solid phase. Two successive intramolecular on-bead cyclizations between a cysteine residue and a chloroacetylated reduced peptide bond are the key steps in the synthesis. The first reduced peptide bond was introduced by a reductive alkylation with a 9-fluorenylmethoxycarbonyl protected amino aldehyde, and the second by coupling of a dipeptide building block containing an allyloxycarbonyl protected reduced peptide bond. The desired bicyclic product was obtained as a mixture of two isomers, which were evaluated for their ability to inhibit the muscular nicotinic acetylcholine receptor expressed in Xenopus oocytes. The two isomers were found to have IC(50) values (inhibitory activities) of 144 microM and 48 microM, compared to 0.18 microM for native conotoxin G1.

The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives were found to be biologically active. In electrophysiological patch-clamp recordings mono-[127I]-BeKm-1 had a concentration of half-maximal inhibition (IC50 value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC50 value of 7 nM. Mono-[125I]-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant ( Kd) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of [(125)I]-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-1); dissociation rate constant, k(off)=0.005 s(-1)). Wild-type BeKm-1 displaced binding of [125I]-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca2+-activated K+-channels, had no effect on [125I]-BeKm-1 binding. Adding the classical rapid delayed rectifier current (IKr) blocker E-4031 reduced binding of [125I]-BeKm-1 to the hERG channel to an IC50 of 7 nM. In autoradiographic studies on rat hearts, binding of [125I]-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.

Ion channels formed by expanded polyglutamine tracts have been proposed to play an important role in the pathological processes leading to neurodegeneration in Huntington's disease and other CAG repeat diseases. We tested the capacity of a huntingtin fragment containing an expanded polyglutamine tract to form ion channels in two cell types. Whole cell current from Xenopus oocytes was recorded using two-electrode voltage-clamp technique, and whole cell current from CHO-K1 cells was recorded by patch-clamp technique. The fragment with an expanded polyglutamine sequence induced no change in the currents recorded in any of the two expression systems, indicating no changes in ion channel activity. The results therefore argue against the proposed hypothesis of expanded polyglutamines forming ion channels.

Activation of KCNQ potassium channels by stimulation of co-expressed dopamine D(2) receptors was studied electrophysiologically in Xenopus laevis oocytes and in mammalian cells. To address the specificity of the interaction between D(2)-like receptors and KCNQ channels, combinations of KCNQ1-5 channels and D(2)-like receptors (D(2L), D(3), and D(4)) were investigated in Xenopus oocytes. Activation of either receptor with the selective D(2)-like receptor agonist quinpirole (100 nM) stimulated all the KCNQ currents, independently of the subunit combination, indicating a common pathway of receptor-channel interaction. The KCNQ4 current was investigated in further detail and was increased by 19.9+/-1.6% ( n=20) by D(2L) receptor stimulation. The effect could be mimicked by injection of GTPgammaS and prevented by injection of Bordetella pertussis toxin, indicating that channel stimulation was mediated via a G protein of the G(alphai/o) subtype. Cells of the human neuroblastoma line SH-SY5Y were co-transfected transiently with KCNQ4 and D(2L) receptors. Stimulation of D(2L) receptors increased the KCNQ4 current ( n=6) as determined in whole-cell patch-clamp recordings. The specificity of the dopaminergic activation of the KCNQ channels was confirmed by co-expression of other neuronal K(+) channels (BK, K(V)1.1, and K(V)4.3) with the D(2L) receptor in Xenopus oocytes. None of these K(+) channels responded to stimulation of the D(2L) receptor. In the mammalian brain, dopamine D(2) receptors and KCNQ channels co-localise postsynaptically in several brain regions, so modulation of neuronal excitability by dopamine release could in part be mediated via an effect on KCNQ channels.

The role of K+ and Cl- channels in salivary secretion was investigated, with emphasis on the potential role of Ca2+ -activated K+ channels. Ligand saturation kinetic assays and autoradiography showed large-conductance (BK) K+ channels to be highly expressed in rat submandibular and parotid glands, whereas low-conductance (SK) K+ channels could not be detected. To investigate the role of K+ and Cl- channels in secretion, intact rabbit submandibular glands were vascularly perfused and secretion induced by 10 microM ACh. Secretion was inhibited by 34+/-3% following perfusion with the general K+ channel inhibitor Ba2+ (5 mM), whereas organic inhibitors of BK (200 nM paxilline) or intermediate-conductance (IK) K+ channels (5 microM clotrimazole) had no effect. Secretion was strongly influenced by Cl- channel inhibitors, as 100 microM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) completely abolished, while 10 microM NPPB, 20 microM NS1652 and 20 microM NS3623 reduced secretion by 34+/-3%, 23+/-3% and 59+/-4%, respectively. In conclusion, although high expression levels of BK channels were demonstrated, pharmacological tools failed to demonstrate any role for BK, IK or SK channels in salivary secretion in the rabbit submandibular gland. Other types of K+ channel, however, and particularly Cl- channels, are essential for ACh-induced salivary secretion.

Ca2+-activated K+ channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were co-expressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca2+ or Cd2+ into the oocyte cytoplasm, before the oocytes were subjected to hyperosmolar or hypoosmolar (+/-50 mOsm mannitol) challenges. In all cases, the oocytes responded rapidly to the osmotic changes with shrinkage or swelling and the effects on the K+ currents were measured. hIK and rSK3 currents were highly sensitive to volume changes and increased immediately to 178% (hIK) or 165% (rSK3) of control in response to swelling and decreased to 64% (hIK) or 61% (rSK3) of control after shrinkage. These responses were dependent on the channels being pre-activated and were almost totally abolished after injection of cytochalasin D into the oocyte cytoplasm (final concentration: 1 microM). In contrast, hBK channels showed only a minor sensitivity to volume changes; the hBK channel activity decreased approximately 20% after swelling and increased approximately 20% after shrinkage. The opposite effects of volume changes on hIK/rSK3 and hBK channels suggest that the significant stimulation of hIK and rSK3 channels during swelling is not mediated by changes in intracellular Ca2+, but rather through interactions with the cytoskeleton, provided that a sufficient basal concentration of intracellular Ca2+ or Cd2+ is present.

Cloned Ca 2+ -activated K + channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca 2+ ] i , evidenced by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling-induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested.

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to −100mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.

KCNE4 is a membrane protein belonging to a family of single transmembrane domain proteins known to have dramatic effect on the gating of certain potassium channels. However, no functional role of KCNE4 has been suggested so far. In the present paper we demonstrate that KCNE4 is an inhibitory subunit to KCNQ1 channels. Co-expression of KCNQ1 and KCNE4 in Xenopus oocytes completely inhibited the KCNQ1 current. This was reproduced in mammalian CHO-K1 cells. Experiments with delayed expression of mRNA coding for KCNE4 in KCNQ1-expressing oocytes suggested that KCNE4 exerts its effect on KCNQ1 channels already expressed in the plasma membrane. This notion was supported by immunocytochemical studies and Western blotting, showing no significant difference in plasma membrane expression of KCNQ1 channels in the presence or absence of KCNE4. The impact of KCNE4 on KCNQ1 was specific since no effect of KCNE4 could be detected if co-expressed with KCNQ2-5 channels or hERG1 channels. RT-PCR studies revealed high KCNE4 expression in embryos and adult uterus, where significant expression of KCNQ1 channels has also been demonstrated.

K+ movement across mitochondrial membranes is involved in volume regulation and may play a role in cardioprotection. The Ca2+-dependent K+ (KCa) channel has been proposed as a contributor to mitochondrial K+ uniport activity, but its functional role is not well understood. To investigate the impact of KCa channels on mitochondrial energetics, we measured K+ fluxes in parallel with ΔΨm and light scattering in isolated mitochondria from guinea pig hearts. We first analyzed the role of different anions on K+ fluxes. Mitochondria loaded with the K+-sensitive fluorescent probe PBFI were incubated with 5mM glutamate-Na+/malate-Na+ in isotonic sucrose medium and subjected to pulses containing different concentrations of KCl, KAc or KH2PO4. K+ fluxes saturated at ≈10mM regardless of the anion, attaining maximal rates (nmol K+/min/mg) of 172±17 (KCl), 84±2.4 (KAc), and 74±3.8 (KH2PO4), with similar K0.5 in all three cases. We then analyzed the effect of NS11021, a novel activator of KCa channels, on the maximal K+ uptake rate. In the presence of KH2PO4 or KAc, 20-50nM of NS11021 increased mitochondrial volume and K+ flux by ∼2.5-fold whereas KCl increased K+ uptake by 30% with little change in volume. ΔΨm was minimally affected in this concentration range. The NS11021 effect was blocked by 200nM charybdotoxin, a KCa channel blocker. At 50nM NS11021, the respiratory control ratio of the mitochondria increased 2.5-fold in the presence of KH2PO4, but not KCl, indicating that a regulatory volume increase is required to improve oxidative phosphorylation. At higher concentrations of the compound (≥1μM) substantial effects on ΔΨm and state 4 respiration were observed, which were not inhibited by Chtx. The findings indicate that activation of K+ fluxes through KCa channels, coupled with swelling without loss of ΔΨm, improves mitochondrial energetic performance.