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Tetrodotoxin blockade on canine cardiac L-type Ca²? channels depends on pH and redox potential.

ABSTRACT: Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na? channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²? current (I(Ca)) in canine cardiac cells. In the present study, the TTX-sensitivity of I(Ca) was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC?? value obtained under physiological conditions) caused 60% ± 2% inhibition of I(Ca) in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 ?M H?O?). Phosphorylation of the channel protein (induced by 3 ?M forskolin) failed to modify the inhibiting potency of TTX; an IC?? value of 50 ± 4 ?M was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels.

SUBMITTER: Hegyi B

PROVIDER: S-EPMC3721225 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Tetrodotoxin blockade on canine cardiac L-type Ca²⁺ channels depends on pH and redox potential.

Hegyi Bence B Komáromi István I Kistamás Kornél K Ruzsnavszky Ferenc F Váczi Krisztina K Horváth Balázs B Magyar János J Bányász Tamás T Nánási Péter P PP Szentandrássy Norbert N

Marine drugs 20130614 6

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na⁺ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²⁺ current (I(Ca)) in canine cardiac cells. In the present study, the TTX-sensitivity of I(Ca) was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fif ...[more]

PMID: 23771047

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Project description:Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

Dataset Information

Tetrodotoxin blockade on canine cardiac L-type Ca²? channels depends on pH and redox potential.

Publications

Tetrodotoxin blockade on canine cardiac L-type Ca²⁺ channels depends on pH and redox potential.

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