ABSTRACT
Oscillations of intracellular Ca2+ provide a novel mechanism for sustained activation of cellular processes. Receptor-activated oscillations are mainly thought to occur through rhythmic IP3-dependent store discharge. However, as shown here in HEK293 cells 1 nM orexin-A (Ox-A) acting at OX1 receptors (OX1R) triggered oscillatory Ca2+ responses, requiring external Ca2+. These responses were attenuated by interference with TRPC3 channel (but not TRPC1/4) function using dominant negative constructs, elevated Mg2+ (a blocker of many TRP channels) or inhibition of phospholipase A2. These treatments did not affect Ca2+ oscillations elicited by high concentrations of Ox-A (100 nM) in the absence of external Ca2+. OX1R are thus able to activate TRPC(3)-channel-dependent oscillatory responses independently of store discharge.
Subject(s)
Calcium Signaling , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , TRPC Cation Channels/metabolism , Calcium Signaling/drug effects , Cell Line , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/pharmacology , Neuropeptides/metabolism , Neuropeptides/pharmacology , Orexin Receptors , Orexins , Receptors, G-Protein-Coupled/agonists , Receptors, Neuropeptide/agonists , TRPC Cation Channels/antagonists & inhibitorsABSTRACT
The molecular mechanisms underlying the regulation of neurotransmission has been an open question for many years. Here, we have examined an interaction between caveolin1 and SNAREs (soluble N-ethylmalemide-sensitive factor attachment protein receptor) which may contribute to the cellular mechanisms underlying changes in synaptic strength. Previously, we reported that application of 4-aminopyridine to hippocampal slices resulted in a persistent potentiation of synaptic transmission and the induction of a short-lasting and specific 40-kDa complex composed of synaptosomal associated protein of 25 kDa (SNAP25) and caveolin1. We have characterized the binding properties of these proteins and observed that in vitro caveolin1 directly associates with both SNAP25 and syntaxin. Caveolin/SNARE interactions are enhanced in the presence of ATP by a mechanism that involves phosphorylation. While caveolin has been associated with cholesterol transport, signal transduction, and transcytosis, this study provides evidence that caveolin is also a SNARE accessory protein.
