Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium

Abstract

When olfactory receptor neurons respond to odours, a depolarizing Cl− efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl − against an electrochemical gradient. In isolated olfactory receptor neurons, the Na + -K + -2Cl − cotransporter NKCC1 is essential for Cl − accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl − efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl − accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl − transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl − transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl − transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl − /HCO 3 − exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na + -Cl − cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl − /HCO 3 − exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl − component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl − accumulation in olfactory receptor neurons.

Ca2+-activated Cl− currents are dispensable for olfaction

“…Hence, previous studies based on 37 have overestimated the role of Ca 2+ -activated Cl -channels. These channels might contribute only about 40% to receptor currents in vivo.The ~40% reduction of fluid-phase EOG in Ano2 -/-mice fits well to the ~39% to ~57% reduction of EOG amplitudes in Nkcc1 -/-mice 11,16 . The Na + K + 2Cl -cotransporter Nkcc1 may accumulate Cl -into OSNs 9-11,13 , a prerequisite for depolarizing Cl -currents.…”

“…It is believed that cytosolic chloride concentration of OSNs is raised by the Na + K + 2Cl -co-transporter Nkcc1 9,10,13 and that [Cl -] is low in the mucus surrounding the cilia 14,15 . However, mucosal ion concentrations are difficult to measure in vivo, and Nkcc1 knockout mice display attenuated electro-olfactograms (EOGs) 11,16 , but normal olfactory sensitivity 17 .To investigate the role of Ca 2+ -activated Cl -channels in olfaction, we disrupted Ano2 in mice. Ano2 is a member of the Anoctamin (Tmem16) gene family, which encodes several Ca 2+ -activated Cl -channels [18][19][20] .…”

“…The superfusate could be switched to a solution containing niflumic acid (NFA). NFA has been widely used to block Ca 2+ -activated Cl -channels in OSNs and other 6 cells 4,6,9,11,16,34,36,37 and inhibits both Ano1 and Ano2 18,20,21,33,34 . The response to odorants was reduced by roughly 40% in Ano2 -/-EOGs compared to the wild type (Fig.…”

“…The Na + K + 2Cl -cotransporter Nkcc1 may accumulate Cl -into OSNs 9-11,13 , a prerequisite for depolarizing Cl -currents. On the basis of the NFA-block of Nkcc1 -/-EOG responses, the authors 11 concluded that a substantial depolarizing Cl -current remained in Nkcc1 -/-OSNs and proposed that other transporters contribute to intracellular Cl -accumulation 11,16 . The sizeable nonspecific component of NFA inhibition observed here, however, suggests that their results 11,16 are consistent with Nkcc1 being the main Cl -accumulator of OSNs.…”

“…It is believed that cytosolic chloride concentration of OSNs is raised by the Na + K + 2Cl -co-transporter Nkcc1 9,10,13 and that [Cl -] is low in the mucus surrounding the cilia 14,15 . However, mucosal ion concentrations are difficult to measure in vivo, and Nkcc1 knockout mice display attenuated electro-olfactograms (EOGs) 11,16 , but normal olfactory sensitivity 17 .…”