Activation of anoctamin 1 is attributed to synergistic effect of voltage and Ca²⁺ in mouse retinal bipolar cells.

Abstract

Purpose : TMEM16A/anoctamin1(ANO1) was recently found in mouse retinal cells to exhibit calcium-activated chloride current (I_CaCC). However, the abundantly expressed ANO1 in bipolar cells was not entirely supported by the electrophysiological profiles, due to the small amplitude and its ambiguous properties (eg. variable Ca²⁺ dependency and whole-cell conductance), and the lack of specific blockers. Thus, we investigates what is the determinant to mask endogenous ANO1 properties and we re-evaluate its capability corresponding to structural evidence in mouse bipolar cells.

Methods : Mice (7~8 week-age, C57BL/6J) were deeply anaesthetized with 4% chloral hydrate. Retinal rod bipolar cells for recording were mainly obtained from enzymatic dissociation and slice preparation was applied for additional experiment. The membrane currents were recorded by the whole-cell patch-clamp technique.

Results : At the dissociated retinal bipolar cells, we found outwardly-rectified ANO1 current in large amplitude >3-folds compared to the normal response. It is strongly Vm-dependent at relatively lower range of [Ca²⁺]_i, showing rapidly activated and long-lasting conductance. However, Ca²⁺-dependent tail current was more prominent at higher range of [Ca²⁺]_i, showing slowly activated and small whole-cell conductance. Ca²⁺-dependent tail current was facilitated by repeated high-depolarization with increasing time, while not influenced by repeated low-depolarization. Such facilitated ANO1 current was little sensitive to Bapta and L-type antagonist, but was considerably diminished by a specific ANO1 blocker, monna. Additionally, in order to investigate contribution of adjacent cells, we examined the suppressive effect of monna in slice retina preparation and found similar blocking effect. Thus, it can be inferred that additional increase of ANO1 tail current is mediated by Vm-dependent activation mode with intrinsic properties.

Conclusions : The present study reveals that endogenous ANO1 of retinal bipolar cells is capable of exhibiting large whole-cell conductance at high voltage ranges, confirming that voltage and Ca²⁺ synergistically contribute to ANO1 enhancement in retinal neuron. Considering the results in the sliced retina, the amplified ANO1 current appears to be closely related to a Vm-dependent mode with intrinsic properties.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.