NAADP signalling in skeletal muscle

Cytoplasmic calcium levels are tightly regulated by second messengers like inositol trisphosphate (InsP3 ) and diacylglycerol. Recently, cyclic ADP-ribose and also nicotinic acid adenine dinucleotide phosphate (NAADP) were established as second messengers that induce Ca2+ release. These findings were originally obtained in sea urchin eggs but analogous observations have been made in mammalian cells such as lymphocytes. Up to now it is not clear which signalling cascade(s) link(s) these second messengers to the rapid Ca2+ efflux from the intracellular stores. Two types of release channels, namely the inositol trisphosphate receptors and the ryanodine receptors, are well characterised candidates. Preliminary experiments from our laboratory provide evidence that the ADP-ribosyl cyclase, the enzyme which generates NAADP, is present in the membranes of the skeletal muscle sarcoplasmatic reticulum and is regulated in a Ca2+ dependent manner. Furthermore, NAADP gates ryanodine sensitive Ca2+ release from these membrane fractions. The goal of the current project is to elucidate the mechanism(s) by which NAADP is synthesised and how it regulates Ca2+ signalling in skeletal muscle. The project intends to verify the working hypothesis that NAADP is a physiological second messenger in skeletal muscle and that its synthesis is therefore subject to regulation by upstream signals. In order to delineate the signalling cascade(s) that lead to the generation and effects of NAADP we shall define: (i) the nature of the ADP-ribosyl cyclase by purification, enzyme kinetics and substrate specificity (ii) the upstream signal(s) leading to ADP-ribosyl cyclase activation (iii) and characterisation of the mechanism of NAADP gated Ca2+ release from skeletal muscle sarcoplasmatic reticulum. It is expected that this work will generate new insights into the physiology of excitation contraction coupling regulated by second messengers.

The intracellular calcium concentration is tightly regulated from protozoa to mammalian cells and essentially regulates functions like fertilization, neurotransmitter and hormone release and muscle contraction. The novel second messengers cyclic ADP-ribose (cADPr) und nicotinic acid adenine dinucleotide phosphate, NAADP both release Ca2+, which was shown for the first time in sea urchin eggs and is now confirmed in cells from plants to mammalians. Although the synthesis of the nucleotides cADPr and NAADP by the family of ADP-ribosyl cyclases is understood on molecular level, it is still a matter of debate which signals control these enzymes. Moreover, an identification of the NAADP target receptor has not been shown. Throughout this project it was demonstrated for the first time that in skeletal muscle NAADP activates the purified ryanodine receptor, an intracellular Ca2+ release channel of the sarcoplasmatic reticulum. The importance of this finding is underlined by the fact that NAADP is synthesized by an ADP-ribosyl cyclase in the same cell compartment i.e. the sarcoplasmatic reticulum, where the ryanodine receptor is enriched. The sarcoplasmatic reticulum represents the essential Ca2+ store for cardiac and skeletal muscle contraction It turned out that the skeletal muscle isoform of the ADP-ribosyl cyclase is distinct from all other yet describe ADP-ribosyl cyclase isoforms with respect to enzymatic characteristics and pharmacological terms. Moreover, a novel channelling mechanism for substrate promiscuity of the ADP-ribosyl cyclase was identified for the co-substrate nicotinic acid. These findings for the first time show that the biggest ion channel in mammalian cells, the skeletal muscle ryanodine receptor has a highly specific regulator, namely NAADP. This may help to understand the role of skeletal muscle ryanodine receptor during physical activity as well as under pathological conditions. Nevertheless, future project will be needed to highlight the role of this novel signalling cascade in health and disease. Noteworthy, it is clear that the proteins involved in this signalling cascade represent novel drug targets and therefore novel therapeutic concepts.