Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

Abstract

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of −261 pA was measured at −50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.

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

“…Previous studies in vomeronasal sensory neurons identified the presence of Ca 2+ -activated currents; some studies found Ca 2+ -activated nonselective cation currents (Liman, 2003;Spehr et al, 2009), whereas other studies found Ca 2+ -activated Cl  currents (Yang and Delay, 2010;Kim et al, 2011;Dibattista et al, 2012). In hamster vomeronasal sensory neurons, Liman (2003) measured Ca…”

“…Vomeronasal sensory neurons were dissociated from the VNO with the enzymatic-mechanical dissociation protocol described in Dibattista et al (2008Dibattista et al ( , 2012, or with the following slight modifications. In brief, the removed vomer capsule was put into a Petri dish containing divalent-free PBS (Sigma-Aldrich) solution where the VNO was extracted and transferred to a tube containing PBS with 1 mg/ml collagenase (type A), incubated at 37°C for 20 min, and transferred twice to Ringer's solution with 5% fetal bovine serum for 5 min.…”

Elevated Cytosolic Cl−Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons

“…-activated Cl − current (Yang and Delay 2010;Kim et al 2011;Dibattista et al 2012). Its molecular correlate has recently been identified as TMEM16A/anoctamin1 (Amjad et al 2015), the founding member of the anoctamin family of Ca 2+ -activated Cl − channels (Caputo et al 2008;Schroeder et al 2008;Yang et al 2008).…”

“…Its molecular correlate has recently been identified as TMEM16A/anoctamin1 (Amjad et al 2015), the founding member of the anoctamin family of Ca 2+ -activated Cl − channels (Caputo et al 2008;Schroeder et al 2008;Yang et al 2008). While different groups have demonstrated expression of both anoctamin1 and anoctamin2 in VSNs (Rasche et al 2010;Billig et al 2011;Dauner et al 2012;Dibattista et al 2012), vomeronasal Ca 2+ -activated Cl − currents were abolished in conditional anoctamin1 null mice (Amjad et al 2015). For interpretation of these results, however, it is essential to determine whether vomeronasal Cl − channels contribute a strongly depolarizing current or, by contrast, if anoctamin1 activation mediates membrane hyperpolarization or shunting inhibition.…”

“…Various models for the actions of either or all products of PLC-dependent lipid turnover have been proposed (Spehr et al 2002;Lucas et al 2003;Zhang et al 2008;Yang and Delay 2010;Kim et al 2011Kim et al , 2012Dibattista et al 2012). These concepts share a common denominator: a key role of cytosolic Ca 2+ elevations and an important, though not indispensable (Kelliher et al 2006;Yu 2015) function of the transient receptor potential (TRP) channel isoform TRPC2 (Liman et al 1999).…”