Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity

TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP2) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP2 also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP2 and inhibits TRPM6. PLC-activation induced PIP2-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx. Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP2, suggesting that they are likely to participate in interactions with PIP2. Our data indicate that PIP2 is required for TRPM6 channel function; hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis

Neurotoxicity and mode of action of N, N-diethyl-meta-toluamide (DEET).

Recent studies suggest that N, N-diethyl-meta-toluamide (DEET) is an acetylcholinesterase inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent. We investigated the mode of action of DEET neurotoxicity in order to define the specific neuronal targets related to its acute toxicity in insects and mammals. Although toxic to mosquitoes (LD50 ca. 1.5 µg/mg), DEET was a poor acetylcholinesterase inhibitor (<10% inhibition), even at a concentration of 10 mM. IC50 values for DEET against Drosophila melanogaster, Musca domestica, and human acetylcholinesterases were 6-12 mM. Neurophysiological recordings showed that DEET had excitatory effects on the housefly larval central nervous system (EC50: 120 µM), but was over 300-fold less potent than propoxur, a standard anticholinesterase insecticide. Phentolamine, an octopamine receptor antagonist, completely blocked the central neuroexcitation by DEET and octopamine, but was essentially ineffective against hyperexcitation by propoxur and 4-aminopyridine, a potassium channel blocker. DEET was found to illuminate the firefly light organ, a tissue utilizing octopamine as the principal neurotransmitter. Additionally, DEET was shown to increase internal free calcium via the octopamine receptors of Sf21 cells, an effect blocked by phentolamine. DEET also blocked Na(+) and K(+) channels in patch clamped rat cortical neurons, with IC50 values in the micromolar range. These findings suggest DEET is likely targeting octopaminergic synapses to induce neuroexcitation and toxicity in insects, while acetylcholinesterase in both insects and mammals has low (mM) sensitivity to DEET. The ion channel blocking action of DEET in neurons may contribute to the numbness experienced after inadvertent application to the lips or mouth of humans

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity

TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP2) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP2 also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP2 and inhibits TRPM6. PLC-activation induced PIP2-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx. Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP2, suggesting that they are likely to participate in interactions with PIP2. Our data indicate that PIP2 is required for TRPM6 channel function; hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis

Validation of an Improved Statistical Theory for Sea Surface Whitecap Coverage Using Satellite Remote Sensing Data

The whitecap coverage at the sea surface is affected by the ratio of kinetic energy to potential energy, &theta;, the wave spectrum width parameter, &rho;, and other factors. This paper validates an improved statistical theory for surface whitecap coverage. Based on the theoretical analysis, we find that the whitecap coverage is more sensitive to &rho; than to &theta;, and the improved statistical theory for surface whitecap coverage is suitable in regions of rough winds and waves. The satellite-derived whitecap coverage data in the westerly wind zone is used to validate the improved theory. The comparison between the results from theory and observations displays a better performance from the improved theory relative to the other methods tested

Effect of phentolamine on the activity of DEET (A), octopamine (B), propoxur (C) and 4-AP (D) on discharge rates of housefly larvae CNS preparations (n = 3–5 preparations per curve, with each concentration replicated 3–5 times).

<p>Data points represent mean percentage increase of baseline firing rate, and error bars represent SEM of drug concentrations replicated at least 3 times. When error bars are absent, it is because they are smaller than the size of the symbol. Data points at each concentration for drug alone were compared to drug + phentolamine, and statistical significance (t-test, P<0.05) is indicated by an asterisk.</p

Effects of DEET on octopaminergic systems in firefly and <i>Sf</i>21 cells.

<p>A) Dose-dependent action of DEET, CDM, and propoxur on the light organ of the firefly, <i>Photinus pyralis</i>. See text for explanation. B) Activation of an octopamine receptor in <i>Sf</i>21 cells shown by internal calcium fluorescence. Bars represent means of normalized fluorescence with error bars denoting SEM, replicated across individual plates of cells (n = 3). Statistics for each column were determined by a paired t-test against matched control raw fluorescent values, where an asterisk represents statistical significance at P<0.05. For labels, OA =  octopamine and PA =  phentolamine. All compounds were applied at 100 µM.</p

Neurophysiological recordings from the CNS of third instar larvae of <i>M.</i> domestica A) Nerve discharges before and after DEET, toluene, and lidocaine treatment across different CNS preparations, as indicated.

<p>Initial firing frequencies in spikes/second (Hz) for each experiment are given to the left of each trace. B) Concentration-response curves for DEET, toluene, and lidocaine on CNS nerve discharge of <i>M. domestica</i> larvae from replicated recordings (n = 4–5 preparations per curve, with each concentration replicated 4 times), as shown in A. Data points represent mean percentage increase of baseline firing rate, and error bars represent SEM of drug concentrations replicated at least 3 times. When error bars are absent, it is because they are smaller than the size of the symbol.</p

AChE inhibition data expressed as mean (n = 3) IC₅₀ values.

<p>Inhibition values of <i>Md</i>AChE, <i>Dm</i>AChE, and hAChE were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Letters after 95% confidence intervals (CI) represent statistical significance for IC₅₀ values among enzymes tested with DEET. IC₅₀s for DEET not labeled by the same letter represent statistical significance (P<0.05).</p

Block of neonatal rat cortical sodium and potassium channels by DEET and related compounds.

<p>A) Inward sodium currents activated by a 15 msec pulse to 0 mV from a holding potential of −80 mV. Control is the peak inward sodium current before treatment. Increasing DEET concentrations blocked the current, each trace taken after 1 min incubation in drug, and there was little change thereafter. Each trace is matched in form to treatment: thick line, Control; thin line, Washout; dashed line, 100 µM DEET; dotted line, 300 µM DEET; and dotted/dashed line, 1 mM DEET. B) Concentration-response curves generated from peak sodium currents as shown in A, replicated across different cells treated with DEET (n = 3), toluene (n = 6), or lidocaine (n = 4). Symbols are mean percentage of control current amplitude, with each concentration of blocker replicated 3–6 times. Error bars represent SEM of currents. C) Typical outward currents activated by a 500 msec pulse to +60 mV from a holding potential of −80 mV, and displayed concentration-dependent block by DEET. D) Typical current-voltage relationships and DEET inhibition of potassium currents in rat cortical neurons. Currents were evoked by stepping the membrane voltage between −100 and +100 mV in 20 mV increments from a holding potential of −80 mV. Amplitude of the sustained current was calculated at 200 msec. E) Concentration-response curves for DEET-mediated inhibition of rat neuronal potassium channels from recordings as shown in C, using responses at +60 mV. Symbols are mean percentage of control current amplitude, and error bars represent SEM of currents replicated across different cells (n≥3).</p