Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca 2+ waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca 2+ via L-SR junctions, in a manner that requires initial Ca 2+ release from lysosomes and subsequent Ca 2+-induced Ca 2+ release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca 2+ signal information as input data. Simulations of NAADP-dependent junctional Ca 2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca 2+ signals and simulated Ca 2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca 2+] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca 2+signal coupling and may contribute to cellular function in health and disease.

This review focuses on how smooth muscle sarcoplasmic reticulum (SR), the major releasable Ca 2+ store in these cells, performs its many functions by communicating with the plasma membrane (PM) and other organelles across cytoplasmic nanospaces, defined by membrane-membrane junctions less than 50 nm across. In spite of accumulating evidence in favour of the view that cytoplasmic nanospaces are a prerequisite for effective control of diverse cellular functions, our current understanding of how smooth muscle cells accomplish site-and function-specific Ca 2+ signalling remains in its infancy. We first present evidence in support of the view that effective Ca 2+ signalling depends on the restricted diffusion of Ca 2+ within cytoplasmic nanospaces. We then develop an evidence-based model of the smooth muscle SR -the 'pan-junctional SR' model -that incorporates a network of tubules and quilts that are capable of auto-regulating their Ca 2+ content and determining junctional [Ca 2+ ] i through loading and unloading at membrane-membrane nanojunctions. Thereby, we provide a novel working hypothesis in order to inform future investigation into the control of a variety of cellular functions by local Ca 2+ signals at junctional nanospaces, from contraction and energy metabolism to nuclear transcription. Based on the current literature, we discuss the molecular mechanisms whereby the SR mediates these multiple functions through the interaction of ion channels and pumps embedded in apposing membranes within inter-organellar junctions. We finally highlight the fact that although most current hypotheses are qualitatively supported by experimental data, solid quantitative simulations are seriously lacking. Considering that at physiological concentrations the number of calcium ions in a typical junctional nanospace between the PM and SR is of the order of 1, ion concentration variability plays a major role as the currency of information transfer and stochastic quantitative modelling will be required to both test and develop working hypotheses.