The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines. (1/32)

The pharmacology of hSK1, a small conductance calcium-activated potassium channel, was studied in mammalian cell lines (HEK293 and COS-7). In these cell types, hSK1 forms an apamin-sensitive channel with an IC(50) for apamin of 8 nM in HEK293 cells and 12 nM in COS-7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC(50)=23 microM), dequalinium (IC(50)=0.4 microM), and the novel dequalinium analogue, UCL1848 (IC(50)=1 nM). These results are very different from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our findings also raise questions about the role of SK1 channels in generating the apamin-insensitive slow afterhyperpolarization observed in central neurones.  (+info)

Compounds that block both intermediate-conductance (IK(Ca)) and small-conductance (SK(Ca)) calcium-activated potassium channels. (2/32)

1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.  (+info)

Photo-induced inactivation of protein kinase calpha by dequalinium inhibits motility of murine melanoma cells. (3/32)

Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKCalpha activity in vitro could be irreversibly inhibited when treated with DECA at low micromolar concentrations and irradiated with 366 nm of light. This approach was used to probe the role of intracellular PKC activity in the motility of metastatic murine melanoma B16 F10 cells and as a target for DECA analogs with increasing PKC inhibitory potencies. Pretreatment of a monolayer of B16 F10 cells with 250 nM of a DECA analog in the presence of UV irradiation for 5 min resulted in 1) complete inhibition of cell motility for up to 4 h in a time-lapse motility assay and 40 to 60% inhibition of cell migration in a Boyden chamber, and 2) inhibition by 40 to 60% of intracellular phosphatidylserine/Ca(2+)-dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKCalpha is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKCalpha was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKCalpha catalytic activity both as a mechanistic component of cell motility and adhesion and as a critical intracellular target of DECA. These studies further suggest that the combined use of UV with nanomolar concentrations of DECA offers an effective chemotherapeutic approach to inhibit metastatic behavior of melanoma cells.  (+info)

Structural mechanisms of QacR induction and multidrug recognition. (4/32)

The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally diverse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.  (+info)

Dequalinium: a novel, high-affinity blocker of CNGA1 channels. (5/32)

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.  (+info)

Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump. (6/32)

Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections. Yet high-resolution structures of ligand transporter complexes have previously been unavailable. We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands. The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding.  (+info)

Small-conductance calcium-activated K+ channels are expressed in pancreatic islets and regulate glucose responses. (7/32)

Glucose-stimulated insulin secretion is associated with transients of intracellular Ca(2+) concentration [Ca(2+)](i) in the pancreatic beta-cell. We identified the expression and function of specific small-conductance Ca(2+)-activated K(+) (SK) channel genes in insulin-secreting cells. The presence of mRNA for SK1, -2, -3, and -4 (intermediate-conductance Ca(2+)-activated K(+) 1 [IK1]) channels was demonstrated by RT-PCR in rodent islets and insulinoma cells. SK2 and -3 proteins in mouse islets were detected by immunoblot and immunocytochemistry. In the tTA-SK3 tet-off mouse, a normal amount of SK3 protein was present in islets, but it became undetectable after exposure to doxycycline (DOX), which inhibits the transcription of the tTA-SK3 gene. The SK/IK channel-blockers apamin, dequalinium, and charybdotoxin caused increases in average [Ca(2+)](i) levels and in frequency of [Ca(2+)](i) oscillations in wild-type mouse islets. In SK3-tTA tet-off mice, the addition of apamin with glucose and tetraethylammonium (TEA) caused a similar elevation in [Ca(2+)](i), which was greatly diminished after DOX suppression of SK3 expression. We conclude that SK1, -2, -3, and IK1 (SK4) are expressed in islet cells and insulin-secreting cells and are able to influence glucose-induced calcium responses, thereby regulating insulin secretion.  (+info)

Cloning and expression of a small-conductance Ca(2+)-activated K+ channel from the mouse cochlea: coexpression with alpha9/alpha10 acetylcholine receptors. (8/32)

Functional interactions between ligand-gated, voltage-, and Ca(2+)-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca(2+)-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea alpha9 and alpha10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca(2+)-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus alpha9/alpha10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the alpha9/alpha10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response.  (+info)

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