Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements.

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10-20% nucleotide deviation from the canonical ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels.

We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.

Direct evidence that scorpion α-toxins (site-3) modulate sodium channel inactivation by hindrance of voltage-sensor movements.

The position of the voltage-sensing transmembrane segment, S4, in voltage-gated ion channels as a function of voltage remains incompletely elucidated. Site-3 toxins bind primarily to the extracellular loops connecting transmembrane helical segments S1-S2 and S3-S4 in Domain 4 (D4) and S5-S6 in Domain 1 (D1) and slow fast-inactivation of voltage-gated sodium channels. As S4 of the human skeletal muscle voltage-gated sodium channel, hNav1.4, moves in response to depolarization from the resting to the inactivated state, two D4S4 reporters (R2C and R3C, Arg1451Cys and Arg1454Cys, respectively) move from internal to external positions as deduced by reactivity to internally or externally applied sulfhydryl group reagents, methane thiosulfonates (MTS). The changes in reporter reactivity, when cycling rapidly between hyperpolarized and depolarized voltages, enabled determination of the positions of the D4 voltage-sensor and of its rate of movement. Scorpion α-toxin binding impedes D4S4 segment movement during inactivation since the modification rates of R3C in hNav1.4 with methanethiosulfonate (CH3SO2SCH2CH2R, where R = -N(CH3)3 (+) trimethylammonium, MTSET) and benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS) were slowed ~10-fold in toxin-modified channels. Based upon the different size, hydrophobicity and charge of the two reagents it is unlikely that the change in reactivity is due to direct or indirect blockage of access of this site to reagent in the presence of toxin (Tx), but rather is the result of inability of this segment to move outward to the normal extent and at the normal rate in the toxin-modified channel. Measurements of availability of R3C to internally applied reagent show decreased access (slower rates of thiol reaction) providing further evidence for encumbered D4S4 movement in the presence of toxins consistent with the assignment of at least part of the toxin binding site to the region of D4S4 region of the voltage-sensor module.

Studies of alpha-helicity and intersegmental interactions in voltage-gated Na+ channels: S2D4.

Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1-S4 transmembrane segments (the "voltage-sensing domain") clustered around a central pore-forming region (S5-S6 segments and the intervening loop). Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4) of sodium channels differs in function from other domains (D1-D3), we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4) of Na(v)1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine). A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h(infinity)-V plots) indicates a substantial amount of alpha-helical structure in S2D4 (peak at 106 degrees, alpha-Periodicity Index (alpha-PI) of 3.10), This conclusion is supported by alpha-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (beta) and exit from (alpha) fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 alpha-helix (N1382 and E1392C) support the existence of electrostatic network interactions between S2 and other transmembrane segments within Na(v)1.4D4 similar to but not identical to those proposed for K+ channels.

An 'Old World' scorpion beta-toxin that recognizes both insect and mammalian sodium channels.

Scorpion toxins that affect sodium channel (NaCh) gating in excitable cells are divided into alpha- and beta-classes. Whereas alpha-toxins have been found in scorpions throughout the world, anti-mammalian beta-toxins have been assigned, thus far, to 'New World' scorpions while anti-insect selective beta-toxins (depressant and excitatory) have been described only in the 'Old World'. This distribution suggested that diversification of beta-toxins into distinct pharmacological groups occurred after the separation of the continents, 150 million years ago. We have characterized a unique toxin, Lqhbeta1, from the 'Old World' scorpion, Leiurus quinquestriatus hebraeus, that resembles in sequence and activity both 'New World'beta-toxins as well as 'Old World' depressant toxins. Lqhbeta1 competes, with apparent high affinity, with anti-insect and anti-mammalian beta-toxins for binding to cockroach and rat brain synaptosomes, respectively. Surprisingly, Lqhbeta1 also competes with an anti-mammalian alpha-toxin on binding to rat brain NaChs. Analysis of Lqhbeta1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying beta-toxin activity. Moreover, Lqhbeta1 resembles beta-toxins by having a weak effect on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs. These multifaceted features suggest that Lqhbeta1 may represent an ancestral beta-toxin group in 'Old World' scorpions that gave rise, after the separation of the continents, to depressant toxins in 'Old World' scorpions and to various beta-toxin subgroups in 'New World' scorpions.