Intracellular chloride ion concentration in differentiating neuronal cell and its role in growing neurite.

Chloride ion (Cl-) is one of the most abundant anions in our body. Increasing evidence suggests that Cl- plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl- concentrations ([Cl-]i) and the length of growing neurites. We measured [Cl-]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl-]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl-]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl-]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl-]i in neurite tip region is crucial for efficient neurite outgrowth.