Abstract
The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes.
Highlights
Calcium-activated chloride channels play an essential role in the physiology of many cell types
Upon restoration of extracellular calcium at 400 seconds, we observed a robust and dramatic increase in calcium entry in cells expressing CLCA2 compared to control cells (Fig 1A, store-operated calcium entry (SOCE); 1D, Peak 2). These cells exhibited an earlier increase in intracellular calcium at 200 seconds, suggesting that CLCA2 might enhance calcium-charging of the ER in addition to enhancing SOCE (Fig 1A, ER; 1D, Peak 1)
CLCA2 regulates intracellular calcium discovered that both CLCA2 and CLCA1 modulate the availability of intracellular calcium
Summary
Calcium-activated chloride channels play an essential role in the physiology of many cell types. ICaCC mediates contraction in response to signaling molecules such as histamine, norepinephrine, and endothelin that stimulate release of intracellular calcium [3]. Despite their obvious physiological significance, the molecular identity of CaCCs was discovered only recently. Two members of the Anoctamin family of multipass membrane proteins, TMEM16A and TMEM16B, were found to mediate a current with the same properties as the classical ICaCC [4, 5, 6, 7]. While TMEM16B is expressed in the central nervous system and implicated in olfactory transduction, TMEM16A is widely expressed in epithelia
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