State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/DeltaKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with beta1 subunits in Hek293t cells. The flecainide-resting block at -140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 micro M when the cell was not stimulated for 1,000 s. At 100 micro M flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an approximately 70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at -140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at -50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 micro M, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 micro M-1s-1 and 12.2 s-1, respectively. Upon repolarization to -140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was approximately 20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.     

Persistent human cardiac Na+ currents in stably transfected mammalian cells

Miniature persistent late Na⁺ currents in cardiomyocytes have been linked to arrhythmias and sudden death. The goals of this study are to establish a stable cell line expressing robust persistent cardiac Na⁺ currents and to test Class 1 antiarrhythmic drugs for selective action against resting and open states. After transient transfection of an inactivation-deficient human cardiac Na⁺ channel clone (hNav1.5-CW with L409C/A410W double mutations), transfected mammalian HEK293 cells were treated with 1 mg/ml G-418. Individual G-418-resistant colonies were isolated using glass cylinders. One colony with high expression of persistent Na⁺ currents was subjected to a second colony selection. Cells from this colony remained stable in expressing robust peak Na⁺ currents of 996 ± 173 pA/pF at +50 mV (n = 20). Persistent late Na⁺ currents in these cells were clearly visible during a 4-second depolarizing pulse albeit decayed slowly. This slow decay is likely due to slow inactivation of Na⁺ channels and could be largely eliminated by 5 μM batrachotoxin. Peak cardiac hNav1.5-CW Na⁺ currents were blocked by tetrodotoxin with an IC₅₀ value of 2.27 ± 0.08 μM (n = 6). At clinic relevant concentrations, Class 1 antiarrhythmics are much more selective in blocking persistent late Na⁺ currents than their peak counterparts, with a selectivity ratio ranging from 80.6 (flecainide) to 3 (disopyramide). We conclude that (1) Class 1 antiarrhythmics differ widely in their resting- vs. open-channel selectivity, and (2) stably transfected HEK293 cells expressing large persistent hNav1.5-CW Na⁺ currents are suitable for studying as well as screening potent open-channel blockers.     

Block of inactivation-deficient cardiac Na(+) channels by acetyl-KIFMK-amide

The Na(+) channel alpha-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na(+) channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2mM elicited a rapid time-dependent block (tau=0.24 ms) of inactivation-deficient human heart Na(+) channels (hNav1.5-L409C/A410W) at +50 mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ms at 1Hz) produced a substantial use-dependent block of both peak and tail currents by approximately 65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na(+) channel inner cavity.     

Chiral aspects of drug action at ion channels: a commentary on the stereoselectivity of drug actions at voltage-gated ion channels with particular reference to verapamil actions at the Ca2+ channel.

Ion channels may be considered as pharmacological receptors possessing specific drug binding sites with defined structure-activity relationships. Accordingly drug binding to ion channels is stereoselective. Interpretation of this stereoselectivity may be complex because of the existence of differences in affinity and access to different channel states. Such state-dependent interactions may give rise to quantitative and qualitative differences in stereoselectivity. The implications of such differences are reviewed for drug action at Na+, K+ and Ca2+ channels. Detailed attention is paid to the actions of verapamil enantiomers in the cardiovascular system where activities differ in vascular and cardiac tissues because of state-dependent interactions and stereoselective first-oass metabolism.     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Specific inhibition of epithelial Na+ channels by antisense oligonucleotides for the treatment of Na+ hyperabsorption in cystic fibrosis

Background

Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl^? secretion and an increased Na⁺ absorption through epithelial Na⁺ channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na⁺ hyperabsorption by inhibiting the expression of the α‐ENaC subunit.

Methods

We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses.

Results

Screening of several AON by co‐injection or sequential microinjection of AON and ENaC mRNA in X. laevis oocytes led to a sustained decrease in amiloride‐sensitive current and conductance. Using primary cultures of human nasal epithelia, we show that AON effectively suppress amiloride‐sensitive Na⁺ absorption mediated by ENaC in CF and non‐CF tissues. In western blot experiments, it could be shown that the amount of ENaC protein is effectively reduced after AON transfection.

Conclusions

Our data comprise an initial step towards a preclinical test with AON to reduce Na⁺ hyperabsorption in CF epithelia. Copyright © 2009 John Wiley & Sons, Ltd.

     

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.     

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production

The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.     

Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer- and blood-brain barrier-related effects

Abstract We have examined whether non-thermal exposures of cultures of the human endothelial cell line EA.hy926 to 900 MHz GSM mobile phone microwave radiation could activate stress response. Results obtained demonstrate that 1-hour non-thermal exposure of EA.hy926 cells changes the phosphorylation status of numerous, yet largely unidentified, proteins. One of the affected proteins was identified as heat shock protein-27 (hsp27). Mobile phone exposure caused a transient increase in phosphorylation of hsp27, an effect which was prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Also, mobile phone exposure caused transient changes in the protein expression levels of hsp27 and p38MAPK. All these changes were non-thermal effects because, as determined using temperature probes, irradiation did not alter the temperature of cell cultures, which remained throughout the irradiation period at 37 ± 0.3 °C. Changes in the overall pattern of protein phosphorylation suggest that mobile phone radiation activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK stress response pathway. Based on the known functions of hsp27, we put forward the hypothesis that mobile phone radiation-induced activation of hsp27 may (i) facilitate the development of brain cancer by inhibiting the cytochrome c/caspase-3 apoptotic pathway and (ii) cause an increase in blood-brain barrier permeability through stabilization of endothelial cell stress fibers. We postulate that these events, when occurring repeatedly over a long period of time, might become a health hazard because of the possible accumulation of brain tissue damage. Furthermore, our hypothesis suggests that other brain damaging factors may co-participate in mobile phone radiation-induced effects.     

Effects of retinoids on the production of tumour necrosis factor-alpha and nitric oxide by lipopolysaccharide-stimulated rat Kupffer cells in vitro: evidence for participation of retinoid X receptor signalling pathway

Kupffer cells play important roles in the development of liver injury by producing cytokines and free radicals. In consequence inhibition of these inflammatory mediators will be one of the targets for treating liver diseases. Retinoids modulate a wide variety of functions of monocytes/macrophages. Cellular effects of retinoids are mediated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We examined the effects of several kinds of natural and synthetic retinoids on the production of tumour necrosis factor-α (TNF-α) and nitric oxide (NO) by LPS-stimulated rat Kupffer cells in vitro. Of the various retinoids tested, 9-cis-retinoic acid (9-cis-RA) and Ro 13-6307, which are agonists of both RARs and RXRs, suppressed the production of TNF-α and NO in a concentration-dependent fashion, whereas three types of RAR-selective agonists, Ro 13-7410, Ro 40-6055 and Ro 19-0645 did not show any effect. Furthermore, the RARα antagonist, Ro 41-5253, did not prevent the effects induced by 9-cis-RA. The results suggest that these effects of 9-cis RA and Ro 13-6307 were induced by the RXRs-dependent signalling pathway. © 1997 John Wiley & Sons, Ltd.