The TAM-associated STIM1 mutation increases ORAI1 channel function due to a reduced STIM1 inactivation break and an absence of microtubule trapping.

Tubular aggregate myopathy (TAM) is a progressive skeletal muscle disease associated with gain-of-function mutations in the ER Ca sensor STIM1 that mediates store-operated Ca entry (SOCE) across the Ca-release-activated (CRAC) Ca channel ORAI1. A frameshift mutation in STIM1 inactivation domain, STIM1, was identified in a TAM patient and reported to decrease SOCE. Using ion imaging and electrophysiology, we show that the STIM1 mutation instead renders STIM1 constitutively active. In ion imaging experiments, STIM1 was less efficient than native STIM1 when expressed alone but enhanced SOCE and increased basal Ca and Mn influx when expressed together with ORAI1. In patch-clamp recordings, STIM1 generated larger pre-activated CRAC currents lacking slow Ca-dependent inhibition (SCDI). STIM1 was pre-recruited in plasma membrane clusters when co-expressed with ORAI1, as were mutants truncated at the frameshift residue or lacking EB-1-binding, which recapitulated STIM1 gain-of-function. When expressed alone in human primary myoblasts, STIM1 was pre-recruited in large clusters and increased basal Ca entry. These observations establish that STIM1 confers a gain of CRAC channel function due to the loss of critical inhibitory C-terminal domains that prevent STIM1 binding to ORAI1, enable STIM1 trapping by microtubules, and mediate SCDI, providing a mechanistic explanation for the muscular defects of TAM patients bearing this mutation.