CFTR Modulates Orai1 Dynamics

Abstract

Calcium signaling regulates many cellular processes and is upregulated in cystic fibrosis (CF) epithelial cells. Agonists stimulate Ca2+ release from ER stores and trigger the formation of a complex between the plasma membrane (PM) Ca2+ channel Orai1 and stromal interaction molecule 1 (STIM1), a primarily endoplasmic reticulum (ER) resident protein which senses Ca2+ store depletion. We studied Orai1-STIM1 complex formation in CFBE epithelial cells expressing either wild-type CFTR (cystic fibrosis transmembrane conductance regulator) or the most common disease-causing mutant ΔF508-CFTR, using temporal image cross-correlation spectroscopy (TICCS). Under control conditions, STIM1 exhibited diffusion (DSTIM1=5.8±0.6 µm2/s) and flow at the PM and ER, respectively, whereas Orai1 exhibited only diffusion at the PM (DOrai1=6.3±2.0 µm2/s). Cross-correlation revealed no interaction between them before depletion of the Ca2+ stores, and their transport was similar in cells expressing wt-CFTR or ΔF508-CFTR. Upon Ca2+ store depletion, STIM1 interacted with Orai1 significantly and both proteins displayed diffusion and flow. The diffusion coefficients of both proteins were significantly reduced (DSTIM1=2.0±0.2 µm2/s, DOrai1=4.1±0.3 µm2/s, DCC= 4.0±0.3 µm2/s), and molecular immobility was significantly increased. Orai1 molecular density was also increased dramatically upon stimulation, consistent with the insertion of Orai1 channels into the membrane. Importantly, this molecular density of Orai1 at the PM was significantly increased in ΔF508-CFTR expressing cells (N=30±4 µm−2) compared to those expressing wt-CFTR (N=21±4 µm−2). We propose a mechanism in which the insertion of Orai1 into the PM during depletion of Ca2+ stores is facilitated by its flow with STIM1 on the ER, and is modulated by wt-CFTR in the PM.