Anna Boccaccio

Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.

Fast Adaptation in Mouse Olfactory Sensory Neurons Does Not Require the Activity of Phosphodiesterase

Vertebrate olfactory sensory neurons rapidly adapt to repetitive odorant stimuli. Previous studies have shown that the principal molecular mechanisms for odorant adaptation take place after the odorant-induced production of cAMP, and that one important mechanism is the negative feedback modulation by Ca2+-calmodulin (Ca2+-CaM) of the cyclic nucleotide-gated (CNG) channel. However, the physiological role of the Ca2+-dependent activity of phosphodiesterase (PDE) in adaptation has not been investigated yet. We used the whole-cell voltage-clamp technique to record currents in mouse olfactory sensory neurons elicited by photorelease of 8-Br-cAMP, an analogue of cAMP commonly used as a hydrolysis-resistant compound and known to be a potent agonist of the olfactory CNG channel. We measured currents in response to repetitive photoreleases of cAMP or of 8-Br-cAMP and we observed similar adaptation in response to the second stimulus. Control experiments were conducted in the presence of the PDE inhibitor IBMX, confirming that an increase in PDE activity was not involved in the response decrease. Since the total current activated by 8-Br-cAMP, as well as that physiologically induced by odorants, is composed not only of current carried by Na+ and Ca2+ through CNG channels, but also by a Ca2+-activated Cl− current, we performed control experiments in which the reversal potential of Cl− was set, by ion substitution, at the same value of the holding potential, −50 mV. Adaptation was measured also in these conditions of diminished Ca2+-activated Cl− current. Furthermore, by producing repetitive increases of ciliarys Ca2+ with flash photolysis of caged Ca2+, we showed that Ca2+-activated Cl− channels do not adapt and that there is no Cl− depletion in the cilia. All together, these results indicate that the activity of ciliary PDE is not required for fast adaptation to repetitive stimuli in mouse olfactory sensory neurons.

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

Extracellular anions more permeant than Cl− modulate TMEM16B gating to promote channel opening, whereas less permeant anions favor channel closure.

Modulation of plant TPC channels by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Vacuolar (SV) channel encoded by the plant TPC1 gene. The patch-clamp technique was applied to isolated vacuoles from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs including the endogenous alpha-linolenic acid (ALA). The addition of micromolar concentrations of AA reversibly inhibited the SV channel decreasing the maximum open probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation.The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the voltage-independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cells.

The phosphoinositide PI(3,5)P 2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P2. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P2 did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P2 elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P2 was applied to hTPC2 expressed in baker’s yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P2-mediated activation and Na+ selectivity of mammalian TPC2.

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

TMEM16A is an essential component of Ca2+-activated Cl− currents in mouse vomeronasal sensory neurons.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

•  Ion transport proteins in intracellular membranes of eukaryotic cells play key roles in many physiological and pathological processes. •  The function of many of these transporters is poorly understood, because their intracellular localization makes them difficult to study. •  Here, we used the large organelle of plant cells, the central vacuole, as a novel system to study an intracellular transporter from animal cells. •  Our data showed that the lysosomal chloride transporter CLC‐7 from rat constitutes a functional transport protein in the central vacuole of the model plant Arabidopsis thaliana (thale cress). •  This novel approach has the potential to elucidate the transport properties of further, poorly studied intracellular ion channels and transporters.

The Ca2+-activated Cl- channel TMEM16B regulates action potential firing and axonal targeting in olfactory sensory neurons

TMEM16B is expressed in olfactory sensory neurons, but previous attempts to establish a physiological role in olfaction have been unsuccessful. Pietra et al. find that genetic ablation of TMEM16B results in defects in the olfactory behavior of mice and the cellular physiology of olfactory sensory neurons.

Tonic and Phasic Tetrodotoxin Block of Sodium Channels with Point Mutations in the Outer Pore Region

Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA alpha-subunit of sodium channels. The various phenotypes are tonically half-blocked at TTX concentrations, IC50(t), that span a range of more than three orders of magnitude, from 4 nM in mutant D1426K to 11 microM in mutant D1717Q. When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time constants that span an equally wide range of values ([TTX] approximately IC50(t), from approximately 60 s for D1426K to approximately 30 ms for D1717Q) and are in most phenotypes roughly inversely proportional to IC50(t). In contrast, all phenotypes show the same approximately threefold increase in their TTX affinity under stimulation. The invariance of the free-energy difference between tonic and phasic configurations of the toxin-receptor complex, together with the extreme variability of phasic block kinetics, is fully consistent with the trapped-ion mechanism of use dependence suggested by and developed by. Using this model, we estimated for each phenotype both the second-order association rate constant, kon, and the first-order dissociation rate constant, koff, for TTX binding. Except for mutant E945Q, all phenotypes have roughly the same value of kon approximately 2 microM-1 s-1 and owe their large differences in IC50(t) to different koff values. However, a 60-fold reduction in kon is the main determinant of the low TTX sensitivity of mutant E945Q. This suggests that the carboxyl group of E945 occupies a much more external position in the pore vestibule than that of the homologous residue D1717.

A Dynamical Feedback Model for Adaptation in the Olfactory Transduction Pathway

Olfactory transduction exhibits two distinct types of adaptation, which we denote multipulse and step adaptation. In terms of measured transduction current, multipulse adaptation appears as a decrease in the amplitude of the second of two consecutive responses when the olfactory neuron is stimulated with two brief pulses. Step adaptation occurs in response to a sustained steplike stimulation and is characterized by a return to a steady-state current amplitude close to the prestimulus value, after a transient peak. In this article, we formulate a dynamical model of the olfactory transduction pathway, which includes the kinetics of the CNG channels, the concentration of Ca ions flowing through them, and the Ca-complexes responsible for the regulation. Based on this model, a common dynamical explanation for the two types of adaptation is suggested. We show that both forms of adaptation can be well described using different time constants for the kinetics of Ca ions (faster) and the kinetics of the feedback mechanisms (slower). The model is validated on experimental data collected in voltage-clamp conditions using different techniques and animal species.