The channel Orai1 requires Ca²⁺ store depletion in the endoplasmic reticulum and an interaction with the Ca²⁺ sensor STIM1 to mediate Ca²⁺ signaling. Alterations in Orai1-mediated Ca²⁺ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca²⁺ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca²⁺ permeation associated with gene transcription and provide a gating mechanism for Orai1.