Tmem16a Ca 2+ Activated Chloride Channels in Sensory Neurons can Discriminate between Different Intracellular Ca 2+ Sources

Abstract

Proteins of Tmem16 (anoctamin) family are the candidate subunits for Ca2+-activated Cl- channels (CaCC). Both Tmem16a expression and CaCC currents were reported in small (predominantly nociceptive) sensory neurons from dorsal root ganglia (DRG). In contrast to CNS neurons, nociceptors accumulate high intracellular Cl- concentration and thus activation of CaCC in these neurons was shown to be excitatory and to contribute to the inflammatory nociceptive responses. We show that CaCC in nociceptors is activated by the release of Ca2+ from the IP3-sensitive intracellular stores in response to bradykinin (BK; 44/47 responsive neurons) or proteases (through PAR-2 receptors; 8/12 responsive neurons). This current was attenuated by 48% by siRNA knock-down of the Tmem16a (n=7). Interestingly, while in the majority of small DRG neurons CACC was induced by Ca2+ release from the stores, only in 12/58 neurons CaCC was activated by the Ca2+ influx through the voltage-gated Ca2+ channels triggered by the voltage pulse. Chelating intracellular Ca2+ with the slow Ca2+ buffer EGTA did not affect CaCC activation by PAR2-AP (5/7 responsive neurons) while the fast buffer BAPTA abolished such activation (1/9 responsive neurons) suggesting a close proximity of the Ca2+ release sites and CaCC. Acute treatment of DRG neurons with lipid raft disrupting agent methyl-beta-cyclodextrin (MBCD) partially restored coupling of CaCC to VGCC but disrupted coupling with the intracellular stores. Thus, after the MBCD treatment activation of VGCC by voltage pulse induced −106.5±51.4 pA CaCC currents in 17/25 neurons while BK and PAR2-AP failed to induce measurable CaCC in 4 and 8 tested neurons respectively (PAR2-AP induced −100±20 pA CaCC in 9/13 MBCD-untreated neurons). Or data suggest that Tmem16a/CaCC channels in small DRG neurons are functionally coupled to the IP3-sensitive Ca2+ stores.