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

Abstract

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 knob͞cilia 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.ion channel ͉ olfaction ͉ olfactory sensory neurons ͉ patch-clamp ͉ sensory coding

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

“…7B). These biophysical properties are similar to those of mBest2 and mBest3 shown previously (30,33,34,41). Eight of ten cells from the same stable line expressed currents of similar size and this is in agreement with the high efficiency of tetracycline induction (47).…”

“…In this study, the Cl Ϫ currents elicited by overexpression of mBest3 were time and voltage independent and displayed only slight outward rectification at the more positive potentials, which is similar to heterologously expressed hBest1, hBest2, dmBest1 (46,49), xBest2a and b (36), and mBest2 (30,34), which exhibited no signs of voltage-dependent activation and inactivation kinetics. This contrasts with ceBest1 (46), hBest4 (49), and in particular, hBest3 (48,49), the human ortholog sharing 83% identity with mBest3, which rectifies inwardly and displays very slow activation kinetics (over a period of many seconds) when elicited by long hyperpolarizing steps.…”

“…Their reported anion selectivity, small unitary conductance, Ca 2ϩ dependence, and sensitivity to Cl Ϫ channel blockers such as DIDS and niflumic acid make this protein a likely molecular candidate for the small conductance Cl Ca expressed in a wide range of cell types including mammalian cardiac myocytes. Three murine Bestrophin homologues (mBest1-3) have been previously identified (20), and the biophysical properties of the current induced by heterologous expression of mBest2 has been studied extensively by two independent groups (30,32,34,35). We successfully recorded Ca 2ϩ -activated Cl Ϫ currents after expressing the full-length mBest3 clone in two different mammalian cell lines by two distinct methods: 1) transient transfection in HEK-tsA201 and COS-7 cells; and 2) stable expression induced by a tetracycline-inducible method.…”

“…In addition, only a few studies have investigated Bestrophin protein expression. Immunohistochemisty studies have localized human Bestrophin 1 to the basolateral membrane of the retinal pigment epithelium (23); murine Bestrophin 1 to tracheal and colonic epithelial cells and kidney (4,5); and murine Bestrophin 2 to the basolateral membranes of olfactory sensory neurons, colonic, tracheal, and nonpigmented cilliary epithelial cells (3,5,30). There have been no studies on the tissue and cellular localization of native Bestrophin 3 in any tissue including the heart.…”

“…A detailed examination of the biophysical properties of mBest2 has been reported (30,32,34,35). Whole cell patch-clamp analysis has also been reported for a truncated form of mBest3 (33,37).…”