Electrochemical strategy to scout 1,4-naphthoquinones effect on voltage gated potassium channels.

Naphthoquinone (NQ) was tested on voltage-gated ion channels expressed in Xenopus laevis oocytes. The activity of potassium Shaker channel with Inactivation domain Removed (ShIR) was not affected; in contrast, NQ diminished Kv1.3 currents. A current decrease was barely observed with the oxidant H(2)O(2). These findings suggested that redox properties were involved in the naphthoquinone-Kv1.3 channel interaction. NQ and some derivatives (NQs) were characterized in DMSO and physiological (ND-96) media by cyclic voltammetry. A typical two-stage mono-electronic reduction mechanism was observed in DMSO, while a one-stage bi-electronic reduction process was found in ND-96 medium. NQs with the lowest and the highest redox potential values were tested on both channels. Voltage-clamp recordings showed that inhibition of Kv1.3 was dependent on NQs redox potential. Results demonstrated that structural features (aromaticity and substituents prone to hydrogen bonds formation) of NQs were also important. This effect could be explained by interactions of some channel residues with NQs that contribute to favor their reduction process in the protein surroundings. The electrochemical strategy presented to simulate the cellular environments (aqueous and non-aqueous) that NQs may face, is an important contribution to pre-select (in a fine and simple way) the best redox compounds for electrophysiological testing.

Functional properties of a truncated recombinant GIRK5 potassium channel.

Xenopus laevis oocytes codify a G-protein-activated inward rectifier potassium channel (GIRK5 or Kir3.5). Coinjection of other GIRKs, the muscarinic m2 receptor, or Gbetagamma protein cRNAs is required to observe functional GIRKx-GIRK5 heteromultimers in oocytes. Studies with GIRK2 isoforms have shown that the size of the amino or carboxyl terminus plays a crucial role on giving functional K(+) channels. In this work we studied the properties of a GIRK5 with 25 amino acids deleted toward its amino-terminal domain. Injection of GIRK5-Delta25 cRNA alone displayed large basal and transient inward rectifying currents in oocytes. The instantaneous currents reached a stationary level after a long duration voltage pulse (10 s). For this relaxation, fast (tau(1)) and slow (tau(2)) time constants were estimated at different voltages. Recovery from inactivation followed a monoexponential function (tau=0.95+/-0.07 s). By contrast with other inward rectifier channels, blockade of GIRK5-Delta25 by extracellular Ba(2+) was voltage-independent (K(d)=102+/-2 microM), suggesting the presence of a Ba(2+) site at the external channel vestibule. To confirm this hypothesis, the Ba(2+) sensitivity of two charged mutants GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) at each of the external loops was determined. GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) showed a 100-fold and 2-fold higher affinity to Ba(2+), respectively, supporting the existence of this Ba(2+) binding site.

Modulation of a calcium-activated chloride current by Maitotoxin.

The effect of Maitotoxin (MTX) on the calcium-activated chloride current (ICl-Ca) from Xenopus oocytes was studied, applying the two-electrode voltage clamp technique. MTX increased the current amplitude at all the voltages explored and reduced the time to reach the maximum current level (time to peak). At low toxin concentrations (15 pM), both effects were fully reversible. Activation of ICl-Ca by MTX was secondary to the increment in the intracellular Ca2+ concentration induced by this toxin, since incubation of the oocytes with the cell-permeant Ca2+ chelator BAPTA-AM, greatly reduced the effect of MTX on ICl-Ca. Furthermore, external chloride ions removal also diminished the MTX effect on the current, strongly suggesting that the main current activated by MTX is ICl-Ca. Subsequent applications of a fixed toxin concentration after toxin washout resulted in enhanced ICl-Ca, suggesting that the toxin effect potentiates.

Maitotoxin, a cationic channel activator.

Maitotoxin (MTX), a water soluble polyether obtained from the marine dinoflagellate Gambierdiscus toxicus is one of the entities responsible for Ciguatera, a form of seafood poisoning. This toxin is a potent activator of changes in the intracellular Ca2+ concentrations of cells from a wide variety of organisms. Evidence published in the last few years strongly suggests that this toxin has no ionophoretic activity. Molecular mechanics studies, shown for the first time in this review, models MTX as a molecular 'wire'. The present work compiles the few studies developed with electrophysiological techniques. All these reports indicate that MTX is activating a voltage independent, nonselective cationic channel, which in some preparations requires the presence of extracellular Ca2+ for channel activation. The conductance estimated from a variety of tissues is in the order of 12-40 pS. Thus far, no specific blocker has been identified for this channel. The nature of the MTX receptor remains a mistery.