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.

Figure 1.

Current responses induced by photorelease of Ca²⁺ in the apical region of mouse vomeronasal sensory neurons. (A) Schematic drawing of a vomeronasal sensory neuron showing the location of application of the UV flash to photorelease Ca²⁺. (B) Whole cell current induced by photorelease of Ca²⁺ at the holding potential of −50 mV. A flash was applied at time t = 0 (indicated by an arrow). (C) Expanded timescale shows the rapid increase in the current upon Ca²⁺ photorelease. The current rising phase was well fitted by a single exponential (red dotted line) with a τ value of 9.5 ms.

Figure 2.

Ion selectivity of the Ca²⁺-activated current. Whole cell currents from vomeronasal sensory neurons induced by photorelease of Ca²⁺ into the apical region recorded at the indicated holding potentials. A UV flash was applied at the time t = 0 (indicated by an arrow). Recordings in the presence of extracellular Ringer’s solution containing 140 mM: (A) NaCl, (B) NMDG-Cl, (C) NaMeS, and (D) NaSCN, each from a different neuron. (E) Average reversal potentials measured in the presence of the indicated ionic solutions: NaCl (n = 6), NMDG-Cl (n = 3), NaMeS (n = 3), and NaSCN (n = 8).

Figure 3.

Blockage of the Ca²⁺-activated Cl⁻ current. Whole cell currents induced by photorelease of Ca²⁺ into the apical region of vomeronasal sensory neurons. The holding potential was −50 mV. Current recordings were obtained before blocker application (control), 2 min after application of the indicated blockers, and 2 min after the removal of blockers (wash). The following blockers were used: (A) 300 µM NFA and (B) 1 mM DIDS. (C) Average values of the current in the presence of 300 µM NFA (n = 5) or 1 mM DIDS (n = 3) normalized to the current in control conditions (P < 0.05).

Figure 4.

Expression of TMEM16s/anoctamins in mouse VNO. TMEM16/anoctamin isoforms A–J (1–10) were amplified from VNO cDNA by RT-PCR. A/1, B/2, F/6, G/7, J/9, and K/10 are expressed in the VNO.

Figure 5.

Specificity of rabbit anti-TMEM16A and anti-TMEM16B antibodies in HEK 293T cells expressing TMEM16A or TMEM16B. (A–F) Fluorescence images of the staining with anti-TMEM16A or anti-TMEM16B antibodies (indicated with the prefix α) of HEK 293T cells transiently cotransfected with TMEM16A and GFP cDNA. Specific staining was observed only with anti-TMEM16A (B), whereas no immunoreactivity was detected with anti-TMEM16B (E) antibody. (G–L) Fluorescence images of the staining with anti-TMEM16B or anti-TMEM16A antibodies of HEK 293T cells transiently cotransfected with TMEM16B and GFP cDNA. Specific staining was observed only with anti-TMEM16B (H), whereas no immunoreactivity was detected with anti-TMEM16A (K) antibody. Cell nuclei were stained by DAPI (blue). Bars, 5 µm.

Figure 6.

TMEM16A and TMEM16B immunoreactivity in the olfactory epithelium. (A–C) Confocal images of coronal sections of the olfactory epithelium from an OMP-GFP mouse showing the absence of TMEM16A immunoreactivity. (D–F) Confocal images of the transition region between the olfactory and the respiratory epithelium (absence of OMP-GFP–expressing neurons). The arrow indicates the transition between the two epithelia. TMEM16B is expressed at the apical surface of the olfactory but not of the respiratory epithelium. Cell nuclei were stained by DAPI (blue). Bars, 20 µm.

Figure 7.

TMEM16A and TMEM16B are expressed at the apical surface of the vomeronasal epithelium. (A–H) Immunostaining of sections of VNO from an OMP-GFP mouse. (A and D) Endogenous GFP fluorescence of mature vomeronasal sensory neurons. TMEM16A (B) and TMEM16B (E) are expressed at the luminal surface of the vomeronasal sensory epithelium. (G and H) High magnification image of the apical portion of the VNO showing that TMEM16A and TMEM16B are expressed at the apical surface. Cell nuclei were stained by DAPI (blue). Bars: (A–F) 20 µm; (G and H) 2 µm.

Figure 8.

TMEM16A and TMEM16B are coexpressed with TRPC2. Double-label immunohistochemistry in tissue sections of VNO from a wild-type mouse showing the coexpression of TRPC2 with TMEM16A (A–C) and TMEM16B (D–F) at the luminal surface of the vomeronasal sensory epithelium. Cell nuclei were stained by DAPI (blue). Bars, 20 µm.

Figure 9.

TMEM16A and TMEM16B are coexpressed in the microvilli of vomeronasal sensory neurons. (A) Bright field image of a vomeronasal sensory neuron isolated from an OMP-GFP mouse (B). The same neuron was stained with (C) rabbit anti-TMEM16A and (D) guinea pig anti-TMEM16B, showing the coexpression of the two anion channels in the microvilli (E). Cell nuclei were stained by DAPI (blue). Bars, 5 µm.

Figure 10.

TMEM16A and TMEM16B are expressed in the microvilli of both apical and basal vomeronasal sensory neurons. TMEM16A (guinea pig antibody) is expressed in the microvilli of both apical neurons, as shown by PDE4A (A–C) immunoreactivity, and basal neurons, labeled with rabbit anti-Gαo antibody (D–F). TMEM16B (guinea pig antibody) is also located in the microvilli of both apical (G–I) and basal (J–L) neurons. Cell nuclei were stained by DAPI (blue). Bars, 5 µm.