Abstract
Purpose: :
Cell volume of trabecular meshwork (TM) cells has been posited to regulate aqueous humor outflow resistance. Regulation of TM cell volume depends on activity of swelling-activated Cl- channels (ICl,swell) whose identity is unknown in these cells. Most tissues express anoctamins Ano1 or Ano2, which are Ca2+-activated Cl- channels (CaCCs). Anoctamins are reported to be components of epithelial ICl,swell, but agreement about their functions and location is incomplete. We are testing whether anoctamins may participate in TM cell volume regulation.
Methods: :
Transformed normal human TM5 and glaucomatous GM3 TM cells and HEK293 cells were studied. Gene expression was measured by reverse-transcription PCR (RT-PCR) and real-time PCR, protein product by western blots, and membrane currents by whole-cell ruptured-patch recording.
Results: :
TM5 cells highly expressed Ano6, but Ano1-2 were not detected (N=3). Ano4 and Ano7-10 were also expressed. HEK293 cells expressed both Ano1 and Ano6, (N=2) whereas GM3 cells expressed Ano2 and Ano6 but not Ano9 (N=2). The Ca2+ ionophore ionomycin (5 micromolar) triggered CaCC currents in TM5 cells, with activating and inactivating currents at depolarizing and hyperpolarizing potentials, respectively. At +100 mV, 5 micromolar ionomycin increased currents from 13 ±4 to 60 ±12 pA/pF. The CaCC blocker tannic acid (100 micromolar) inhibited these CaCC currents by 78 ±4% (N=3). Knockdown (75%) of Ano6 markedly reduced ionomycin-activated currents to 12 and 19 pA/pF in duplicate experiments. ICl,swell after 20% hypotonicity reached 188 ±40 pA/pF from 8 ±3 pA/pF. The CaCC inhibitors CaCCinh-A01 (50 micromolar) and tannic acid (100 micromolar) inhibited ICl,swell by 45 ±5% and 96 ±1%, respectively (N=4), but the selective inhibitor of Ano1 and Ano2, T16Ainh-A01 (20 micromolar), was ineffective (N=3).
Conclusions: :
Transformed normal TM cells did not express the best characterized channels of CaCCs, Ano1-2, but highly expressed Ano6, and this expression differed from that of transformed glaucomatous TM cells. The early data suggest that Ano6 may play a role in CaCCs, and that anoctamins may modulate ICl,swell and thereby TM cell volume regulation.
Keywords: outflow: trabecular meshwork • ion channels • calcium