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E-Conference: Defining Calcium Entry Signals

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Lipid Rafts- Hotspots for agonist mediated calcium entry?

2 June 2004

Randen L. Patterson

There are more than 3000 G-protein and receptor tyrosine kinase coupled receptors encoded by the human genome. Although not all of these receptors are encoded by all cells, each cell does contain numerous receptors, even specialized cells such as B and T cells. This observation alone predicts that micro-domains of calcium entry must exist. To obtain specificity of receptor responses for cellular functions, it is logical that the cell must segregate the receptor response into discreet areas, to grant the receptor a specific response.

Evidence to support this hypothesis is rapidly increasing. Some of the most convincing work comes from Delmas et al (Neuron 2002), using TRPC channels as biosensors to test muscarinic receptors and bradykinin receptors. Not surprisingly, each receptor demonstrated a functionally distinct output. We also observed functionally distinct receptor specific calcium entry responses when studying RACK1 interactions with the IP3- receptor. The work from other laboratories, in particular Ambudkar’s group at NIH, provide evidence for spatially restricted expression of TRPC channels into lipid raft/caveolar/actin-rich domains which contain many of the proteins implicated in agonist-mediated calcium entry (PLC, SERCA, IP3 -receptors, calmodulin, heterotrimeric G-proteins, scaffolding proteins etc.).

I think the question is not whether calcium entry occurs within micro -domains, rather how do we investigate these domains? How do we determine the constituents of these regions within the cell? Although lipid rafts are still loosely defined, biochemical methods have been developed to isolate and enrich these areas for study. One of the most important areas of study which has been largely overlooked in the calcium entry field IS trying to determine the molecular environment around TRP channels. What are the other scaffolding proteins tethering these channels? Which other effecter molecules are binding to this complex? Biochemical isolation of TRP channel complexes from discreet regions of the cell, coupled to mass spectrometry sequencing would provide invaluable information as to the molecular environment in which these channels exist.

Why would TRP channels localize to these regions? Lipids, in particular PIP2 whose levels can be rapidly regulated via receptor stimulation, regulate the activity of a wide variety of channels and exchangers in the plasma membrane, from Na/H exchangers to potassium channels (which are found in lipid rafts). Lipids have been demonstrated to modify the function of TRPC 3, 6, 7, in addition to the TRPV and TRPM families. It seems reasonable these channels would localize to areas where lipid organization could be tightly controlled. TRPV and TRPM family proteins can be regulated by PIP2, but the TRPC channels regulation by lipid is still a mystery. TRPC 3, 6, 7 can be activated by the application of exogenous DAG analogues in overexpression systems, although endogenous calcium channel activity observed by the application of exogenous DAG analogues has been observed in only a few reports. As direct lipid binding to any of these channels has yet to be demonstrated, this seems a reasonable place to begin addressing this question. Determination of the lipids for which these channels have affinity will provide information not only about their activity, but likely their trafficking as well.

All of this is not to say that functional TRP channels do not exist outside of lipid rafts, but lipid rafts provide a functional micro-domain which can be focused on, and then expanded to other regions of the plasma membrane.

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Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882