Evolutionarily conserved hydrophobicity and sterics in TM3 / TM4 balance Orai1 pore opening

The calcium (Ca 2+ ) channel Orai1—the pore‐forming subunit of the Ca 2+ release‐activated Ca 2+ channel—is opened by its physiological activator, the endoplasmic reticulum (ER)‐resident Ca 2+ sensor stromal interaction molecule 1 (STIM1) upon ER Ca 2+ store‐depletion. STIM1 primarily engages the Orai1 C‐terminus, relaying an activation signal from the channel periphery via all transmembrane (TM) domains to the Ca 2+ ‐conducting pore. We previously demonstrated that STIM1 coupling elicits conformational changes in the Orai1 TM3/TM4‐interface inducing pore opening. Here, we reveal that a subtle balance of steric and hydrophobic interactions around V181 (TM3), F250 and F253 (TM4) is required for proper physiological function. We show that side chain volume and hydrophobicity in this region jointly determine not only the stability of the channel complex in the closed state, but also the extent of Ca 2+ influx. In physiological settings, this fine‐tuning around V181 permits sufficient activation, while preventing excessively large Ca 2+ current.