Guylain Boulay, Darren M. Brown, Ning Qin, Meisheng Jiang, Alexander Dietrich, Michael Xi Zhu, Zhangguo Chen, Mariel Birnbaumer, Katshuhiko Mikoshiba, and Lutz Birnbaumer
PNAS | December 21, 1999 | 96:26 | 14955-14960

Presentation by: Jordan B. Sylvester

Abstract

Homologues of Drosophilia transient potential receptor (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca2+ stores.  In agreement with this hypothesis, cells expressing TRPs display novel Ca2+-permeable cation channels that can be activated by the inositol 1,4, 5-triphosphated receptor (IP3R) protein.  Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells in still missing.  Here, we document physical interaction of TRP and IP3R by coimmunopreciptation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7.  These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca2+ entry and to test for a role of their respective binding partners in Ca2+ entry.  C7 and an F2q-containing fragment of IP3R decreased both forms of Ca2+ entry.  In contrast, F2g enhanced the two forms of Ca2+ entry.  We conclude that store depletion-activated Ca2+ entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs.  IP3Rs, therefore, have the dual role of releasing Ca2+ from stores and activating Ca2+ influx in response to either increasing IP3 or decreasing luminal Ca2+.
 

My Summary

The intracellular concentration of the IP3 molecule can be elevated by stimulation of certain receptors on the plasma membrane.  There are two resulting effects of such an increase:  (1) Ca2+ is released from the internal stores via the IP3R and (2) Ca2+ enters the cell via ubiquitous store-operated calcium entry (SOCE) channels.  The net result is an increase in the levels of cytoplasmic Ca2+.

Mounting evidence suggests that the major structural components of SOCE channels are TRP proteins.  TRP proteins were originally found in the eyes of Drosophilia melanogaster and their name refers to the transient receptor potential created in the cells mutants that lack the protein. Currently, seven unique TRP homologues have been characterized in numerous mammalian species, including: mice, rats and humans.

Despite all of the work that has been done on TRP channels, it is still not clear how they are operated.  Three models exist:

1. Diffusible Messenger:  Store-depletion causes the release of a "Ca2+ influx factor" into the cytoplasm  This factor then travels to the SOCE channel causing the Ca2+ flood-gates to open, if you will.  Many potential messengers have been suggested, including: cGMP, IP3, diacylglycerol, G-protein, arachidonic acid derivatives, and Ca2+.

2. Channel insertion:  When the intracellular stores are Ca2+ depleted, vesicles containing SOCE channels are exocytotically inserted into the plasma membrane.  I have yet to see evidence of SOCE channel insertion.

3. Conformational Coupling:  This model involves direct, short-range protein-protein interaction between the IP3R on the endoplasmic reticulum and the SOCE channel on the plasma membrane.  Previous work showed that Ca2+ entry channels in HEK cells which stably expressed TRP3 could be activated in inside-out membrane patches by adding either IP3R-rich microsomes and liposomes carrying IP3Rs lacking the C-terminus.
 

This paper gives convincing evidence for the latter model, including:  coimmunoprecipitation of TRP and IP3R, in vitro interactions of various IP3R and TRP fragments, and the functional relevance of these in vitro-interacting sequences on store mediated Ca2+ entry.

Coimmunoprecipitation of TRP and IP3R (Figure 1):

Procedure:
Cell lysates were obtained from HEK cells which stably expressed either HA-TRP3, myc-TRP or HA-TRP6.  Detergent extracts were incubated with the appropriate Ab and subsequently bound to protein A-Sepharose beads.  These beads were collected by centrifugation and washed.  The precipitate and supernatant were run on SDS/PAGE gels and Western blotted with the appropriate Ab.

These Western blots clearly showed that IP3R-3 co-immunoprecipitates specifically with TRPs 3 and 6, and was not the a result of non-specific trapping.  Moreover, IP3R-3 binding appears to be specific for mature TRP molecules since only those extracts which were deglycosylated  were visible by Western blot.

In vitro interactions of IP3R and TRP (Figures 2 & 4):

Procedure:
cDNA fragments of IP3R (F1, F2, etc…) or TRP3 (NT, C1, etc…) in a transcription competent vector were in vitro translated with [35S]-methionine and [35S]-cysteine.  The transcription products were incubated with a GST-fused-fragment of the opposite protein (ie. TRP with IP3R and vice versa) and adsorbed to glutathione (GSH) Sepharose beads.  The sepharose beads were washed and the adsorbed fragment complexes were run on SDS-PAGE gels.

The initial experiments indicate that the F2 region of the IP3R-3 molecule binds to the C-terminal fragment of TRP3 (C7).  More specific analysis of this binding specifies two particular regions of binding: F2q (30 amino acids) and F2g (71 amino acids).

Functional Relevance of the in vitro-interacting sequences (Figures 5 & 6):

Procedure:
IP3R-interacting TRP fragment (C7) and TRP-interacting IP3R fragments (F2l and F2g) fused to EBFP (enhanced blue flourescence protein) were translated into HEK-293T cells, along with the Gq-coupled V1a receptor.  Cells were fura 2-loaded prior to the initial time point.  Cells were bathed in EGTA, exposed to either AVP (which stimulates V1a receptor) or thapsigargin, and then Ca2+ was re-introduced.

The net result here was that both means of stimulating SOCE were affected by the introduction of IP3R-3-inteactioning TRP3 fragments and vice versa.  The TRP3 C7 fragment and the IP3R-3 F2l fragment (which houses F2q) both deminished thapsigargin induced and IP3 stimulated SOCE, while F2g stimulated SOCE.
 

 The Take Home Message:

These experiments show, for the first time, the coimmunoprecipitation TRP with IP3R, the specific sites of interaction on each molecule and, finally, show a significant relationship between these sites and SOCE.  Since the two TRP3-binding IP3R-3 sequences are within 50 amino acids of each other and that they appear to have such opposite effects on SOCE, the relative proximity makes the idea of an on-off switch rather appealing.  Such a model has F2g opening TRP3 channels in response to store-depletion, while F2q closes the channels when the stores are full.  This model by no means negates neither the “Diffusible Factor” model nor the “Channel Insertion” model, rather, it verifies that the “Conformational Coupling” model exists in various cellular systems.
 
 

Follow-up Questions

1. Can this type of coimmunoprecipitation be seen for all forms of IP3Rs and TRPs?

2. Are IP3Rs constitutively bound to TRPs or do they only bind in response to store depletion?

3. What if conformational coupling is not possible due to distance?  Are SOCE channels only responsive to PM localized intracellular Ca2+ stores or does the “Diffusible Factor” model work in cases where distance is a problem?  If so, which has a more important role in SOCE?