A non-transmembrane channel formed by Ca²⁺-bound calsequestrin-2: Calcium Signaling and Excitation–Contraction in Cardiac, Skeletal and Smooth Muscle

Although it is well known that ion channels conduct ions across biomembranes, whether ions are conducted by some non-membrane proteins is not known because of the lack of a detection method. Calsequestrin-2 (CSQ2) is a sarcoplasmic reticulum (SR) Ca²⁺-binding protein suppling Ca²⁺ for the ryanodine receptor Ca²⁺ release during the excitation–contraction coupling in cardiomyocytes. CSQ2 mutations, even in some heterozygous occasions, causes catecholaminergic polymorphic ventricular tachycardia (CPVT2), suggesting that CSQ2 may function beyond a Ca²⁺ buffer. Here, we identify a non-transmembrane channel in Ca²⁺-enriched CSQ2 dimers, which facilitates fast Ca²⁺ mobilization. Using crystallography, we solved the high-resolution structure of Ca²⁺-bound CSQ2 and discovered that the negatively charged residues at the dimer interface encompassed a tubular channel-like structure, dubbed “tunnel,” in which ∼15 Ca²⁺ ions aligned across the ∼5 nm tunnel path. To determine the potential tunnel conductance, we developed a graphene-based nanoelectronic technology to connect a CSQ2 dimer into a nanocircuit. In the Tyrode solution containing 1 mM Ca²⁺, a CSQ2 dimer exhibited a conductance one order of magnitude higher than the background level. This conductance was Ca²⁺ dependent, and was largely suppressed by the single-residue mutation D309N at the bottleneck region of the tunnel path, indicating that the tunnel conducted Ca²⁺ fluxes. When the D309N mutant CSQ2 was expressed in wild-type rat cardiomyocytes by adenoviral vectors, isoproterenol treatment induced chaotic Ca²⁺ waves, delayed after-depolarizations and trigged activities resembling those occurring in CPVT2 models. This dominant negative effect of CSQ2 mutation agreed well with our structural observation that CSQ2 tunnels were interconnected to form a tunnel network. Taken together, these results revealed that CSQ2 builds a nano-highway network for energy-efficient Ca²⁺ mobilization in the SR. Factors that block the Ca²⁺ highway may lead to arrhythmogenesis.