Lipid rafts/caveolae as microdomains of calcium signaling

Abstract

Ca(2+) is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca(2+) that is achieved by the activation of several plasma membrane (PM) Ca(2+) channels as well as release of Ca(2+) from the internal stores. One such channel is referred to as store-operated Ca(2+) channel that is activated by the release of endoplasmic reticulum (ER) Ca(2+) which initiates store-operated Ca(2+) entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca(2+) signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca(2+) signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca(2+) channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca(2+)-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca(2+)-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.

Figure 1. Model depicting major components of cellular Ca²⁺ homeostasis

A prime signaling cascade that mediates changes in [Ca²⁺]_cyt (cytosolic) initiates with the activation of plasma membrane (PM) receptors (ex-GPCR, G-protein coupled receptor). This leads to generation of the diffusible messenger - IP₃ (inositol 1,4,5-trisphosphate) which releases the [Ca²⁺]_ER by activating IP3R (IP3 receptor). Depletion of the ER/SR(endoplasmic/sarcoplasmic reticulum) stores induces clustering of STIM proteins (stromal interaction molecule1 and 2) and the STIM1 clusters/puncta then facilitate Ca²⁺ influx via the store operated Ca²⁺ channels. Besides this other significant Ca²⁺ influx routes such as, receptor operated (ROCC), voltage gated (VGCC), ligand gated (LGCC), and second messenger regulated (SMCC) calcium channels also contribute to increase in [Ca²⁺]_cyt. Other components that are also known to regulate [Ca²⁺]_cyt include - undefined (?) source such as Ca²⁺ leaks/diffusion, ER translocon and debatable components such as CIF (Ca²⁺ influx factor) and PM IP3R. In addition to Ca²⁺ buffering by proteins such as calmodulin, calreticulin etc., the steady-state levels of [Ca²⁺]_cyt is achieved by its extrusion into cells exterior and/or by sequestration into organelles. The major components that bring about this homeostasis of Ca²⁺ includes Ca²⁺ ATPases of the PM (PMCA), ER (SERCA pump), golgi (SPCA – secretory pathway Ca²⁺ ATPase), nucleus (NPC - nuclear pore complex), NCX (sodium Ca²⁺ exchangers), VDAC (voltage dependant anion channel) and the mitochondrial uniporter. The PM Ca²⁺ receptor (CaR) is known to act via sensing the extracellular [Ca²⁺] whereas the mitochondrial permeability transition pore complex (PTP) regulates the change in mitochondrial membrane potential (Δψ) in response to mitochondrial [Ca²⁺]. In aggregate, the resultant increase in [Ca²⁺]_cyt, via various PM Ca²⁺ channels, would engage (to list a few) a variety of short-term (ex-phosphorylation, secretion) and long-term (ex-gene regulation, proliferation, death) cellular functions thereby translating extracellular cues into observable physiological changes. Other abbreviations: RTK – receptor tyrosine kinase, PLC – phospholipase C, PIP2 – phosphatidylinositol 4,5-bisphosphate, DAG – diacylglycerol, PLN –phospholamban, NFAT (nuclear factor of activated T cells), NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells).

Figure 2. Lipid rafts/caveolae in salivary epithelial cells

(A) Confocal image of human submandibular gland (HSG) cells stained for endogenous caveolin1. (B) Transmission electron micrograph (TEM) of HSG cells indicating caveolar microdomains and (C) enlarged section from (B) showing caveolae (omega ‘Ω’ shaped membrane invaginations). (D) A model indicating various components of caveolae. (E) Representative blots of caveolae/lipid raft associated proteins, obtained from HSG cells by floatation gradients as described in [20]. Abbreviations: Mt – mitochondria, PTRF (polymerase I transcript releasing factor), Cav (Caveolin 1, 2), Flot (flotilin 1, 2)

Figure 3. TRPC – topology, Cav1 binding motif and lipid raft association

(A) Model showing the topology of TRPC channels with N- and C-termini regulatory motifs and examples of proteins that are known to interact with TRPC channels is also listed. (B) Partial N- and C-termini sequence alignment of TRPCs indicating putative Cav1 binding motif. Amino acids shown in red are the aromatic residues that are predicted be involved in TRPC-Caveolin interactions. Amino acids shown in green are critical for STIM1 gating of the TRPC channel [28] and are shown here to overlap with the C-terminus Cav1 binding sequence. (C) Presence of TRPC channels in raft/non raft fractions isolated from HSG cells. Lipid rafts were isolated as described in [20] from HSG cells expressing HA/FLAG tagged TRPC cDNAs. Western blotting of the fractionated samples was performed with anti-tag antibodies. Endogenous Cav1 was used as marker for raft fraction, whereas transferrin receptor (TfR) was used as a non-raft marker.