Ca²⁺- and Volume-sensitive Chloride Currents Are Differentially Regulated by Agonists and Store-operated Ca²⁺ Entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca²⁺]_i in a Ca²⁺-free medium followed by a secondary [Ca²⁺]_i rise upon Ca²⁺ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca²⁺-activated (I_Cl(Ca)) and volume-regulated (I_{Cl, swell}) chloride currents. NPPB and DIDS more efficiently inhibited I_Cl(Ca) and I_{Cl, swell}, respectively. Cell swelling caused by hypotonic solution invariably activated I_{Cl, swell} while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced I_{Cl, swell}, while its inactive analogue U-73343 had no effect. I_Cl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca²⁺]_i in the pipette solution at 500 nM. In contrast, I_{Cl, swell} could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP₃ infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I_{Cl, swell} in a nonadditive manner, suggesting their convergence on a common pathway. I_{Cl, swell} and I_Cl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I_Cl(Ca), but abolished I_{Cl, swell}, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I_{Cl, swell} can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a “selection” switch between closely localized channels.