Excitation-contraction coupling in skeletal muscle requires the release of intracellular calcium ions (Ca ²⁺ ) through ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. Half of the RyR1 channels are activated by voltage-dependent Ca ²⁺ channels in the plasma membrane. In planar lipid bilayers, RyR1 channels exhibited simultaneous openings and closings, termed “coupled gating.” Addition of the channel accessory protein FKBP12 induced coupled gating, and removal of FKBP12 uncoupled channels. Coupled gating provides a mechanism by which RyR1 channels that are not associated with voltage-dependent Ca ²⁺ channels can be regulated.

—Excitation-contraction coupling in heart muscle requires the activation of Ca ²⁺ -release channels/type 2 ryanodine receptors (RyR2s) by Ca ²⁺ influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed “coupled gating.” Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca ²⁺ release.

Significance: Reactions of sulfur-centered nucleophiles with nitrogenous species have been studied independently for more than a century for synthetic/industrial purposes; to understand geochemical, atmospheric, and biological processes; and to explain the origins of life. Various products and reaction mechanisms were proposed. We here identify a singular process comprising a network of cascading chemical reactions that form three main bioactive products at physiological pH: nitrosopersulfide, polysulfides, and dinitrososulfite. These anionic products scavenge, transport, and release NO/HNO or sulfide/sulfane sulfur, each displaying distinct chemistries and bioactivities. Our observations provide a chemical foundation for the cross-talk between the NO and H₂S signaling pathways in biology and suggest that the biological actions of these entities can be neither considered nor studied in isolation.

Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca ²⁺ ] _i ) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP ₃ ). IP ₃ activates the IP ₃ receptor (IP ₃ R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP ₃ R, which induced tyrosine phosphorylation of the IP ₃ R. Fyn activated an IP ₃ -gated calcium channel in vitro, and tyrosine phosphorylation of the IP ₃ R during T cell activation was reduced in thymocytes from fyn ^−/− mice. Thus, activation of the IP ₃ R by tyrosine phosphorylation may play a role in regulating [Ca ²⁺ ] _i .

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.