ORAI Channels in Vascular Smooth Muscle

This chapter describes how to study the activities of the ORAI channels in synthetic Vascular Smooth Muscles (VSMCs). Using optical and electrophysiological methods, ORAI channel activity is essentially undetected in quiescent VSMCs but becomes prominent in synthetic VSMCs. In quiescent VSMCs, the expression of ORAI and STIM proteins is either very low or absent and pharmacological store depletion failed to activate store-operated Ca²⁺ entry (SOCE). In contrast, ORAI1 and STIM1 are upregulated in synthetic VSMCs, which are found in diseases of vessel remodeling. Atomic resolution structures of mammalian ORAI isoforms are currently undefined, but based on the drosophila structure and studies using concatenated oligomers of ORAI1, functional mammalian ORAI channels are likely hexamers. The SOCE current mediated by ORAI1 channels is termed the Ca²⁺ release-activated Ca²⁺ current. The most common Ca2+ indicator used to measure global cytosolic Ca²⁺ signals generated by the SOCE pathway is the indicator Fura2, originally developed by RY Tsien and collaborators.