Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel.

Ca(2+)-activated Cl(-) currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl(-) channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13µM Ca(2+). However, in the presence of 1.5µM Ca(2+) (but not in 13µM Ca(2+)), A9C also induced a strong potentiation of tail currents measured at -100mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.

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

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca(2+)-activated Cl(-) channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl(-) with other anions (PX/PCl) was SCN(-) > I(-) > NO3 (-) > Br(-) > Cl(-) > F(-) > gluconate. When external Cl(-) was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca(2+) were modified according to the sequence of permeability ratios, with anions more permeant than Cl(-) slowing both activation and deactivation and anions less permeant than Cl(-) accelerating them. Moreover, replacement of external Cl(-) with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl(-) with SCN(-) shifted G-V to more negative potentials. Dose-response relationships for Ca(2+) in the presence of different extracellular anions indicated that the apparent affinity for Ca(2+) at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca(2+) in the presence of intracellular SCN(-) also increased compared with that in Cl(-). Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.

Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure-function study for this channel.

Inhibitory potential of ginger extracts against enzymes linked to type 2 diabetes, inflammation and induced oxidative stress.

Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Gingerols, the major pungent components of ginger, are known to improve diabetes, including the effect of enhancement against insulin sensitivity. In the current study, ginger sequentially extracted with different solvents-namely, hexane, ethyl acetate, methanol, 70% methanol-water and water-were screened to determine the variations in phenolic-linked active constituents. The potential of these extracts to inhibit key enzymes relevant to type 2 diabetes and inflammation was studied. Phenolic compounds-namely, gingerols and shoagols-were quantified using high-performance liquid chromatography. Ethyl acetate extract showed higher activity compared with other extracts. These studies indicate that ginger has very good potential for α-glucosidase and α-amylase inhibition relevant for type 2 diabetes management and cyclooxygenase inhibition for inflammation.