Coassembly of Trp1 and Trp3 Proteins Generates Diacylglycerol- and Ca2+-sensitive Cation Channels

Abstract

To analyze the functional consequences of coassembly of Trp1 and Trp3 channel proteins we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…The precise mechanism between PKC activation and TRPC3 opening remains however to be firmly established, but our data suggested that Src is, at least partially, involved in this process. Considering that the inhibition of the Ca 2+ entry varied between about 40 and 60% upon src inhibition or siRNA treatment against PKC , it is possible that for a part, TRPC3 is also getting directly activated by DAG, like it was already reported [15,63,64]. This point, however, remains to be further investigated.…”

TRPC3: a versatile transducer molecule that serves integration and diversification of cellular signals

“…The concept implies that a given TRPC species is able to associate with other channel and auxiliary subunits to form distinct signaling complexes with a composition dependent on the availability of the complex partners. Potential signaling partners, as identified in functional and protein-protein interaction assays, are other TRPC species (Lintschinger et al 2000;Goel et al 2002;Hofmann et al 2002) as well as scaffold and ion transport proteins (Kiselyov et al 1999;Rosker et al 2004). It is expected that the oligomerization with pore-forming proteins, e.g., other TRPC species, will change the biophysical and pharmacological properties of the permeation pathway, while association with scaffolds and signaling partners that do not contribute to the TRPC pore may alter other features such as activation and coupling to specific cellular functions.…”

“…In analogy to the architecture of voltage-gated K + channels, formation of a pore structure is assumed to require assembly of TRPC3 in a homo-or heterotetrameric complex with four short hydrophobic segments each located between transmembrane segments five and six of the individual subunits lining the central ion conducting pathway (reviewed in Clapham et al 2001). Overexpression of this protein in classical expression systems such as HEK293 and CHO cells was found to generate a cation conductance with unobtrusive biophysical properties being essentially nonselective (pCa/ pNa about 1.5; Kamouchi et al 1999), and fairly voltage independent (Zitt et al 1997;Hurst et al 1998;Lintschinger et al 2000;Philipp et al 2003). One glimpse of a signature detectable in whole cell TRPC3 currents is the rather noisy appearance at negative membrane potentials (Hurst et al 1998;Lintschinger et al 2000), indicative of a rather large unitary conductance of these channels.…”

“…Overexpression of this protein in classical expression systems such as HEK293 and CHO cells was found to generate a cation conductance with unobtrusive biophysical properties being essentially nonselective (pCa/ pNa about 1.5; Kamouchi et al 1999), and fairly voltage independent (Zitt et al 1997;Hurst et al 1998;Lintschinger et al 2000;Philipp et al 2003). One glimpse of a signature detectable in whole cell TRPC3 currents is the rather noisy appearance at negative membrane potentials (Hurst et al 1998;Lintschinger et al 2000), indicative of a rather large unitary conductance of these channels. Indeed, single TRPC3 channels generated by heterologous overexpression have repeatedly been characterized and a unitary conductance of 60-66 pS (Zitt et al 1997;Hurst et al 1998;Kamouchi et al 1999) as well as a 17 pS subconductance (Kiselyov et al 1999) have been reported.…”

“…Indeed, single TRPC3 channels generated by heterologous overexpression have repeatedly been characterized and a unitary conductance of 60-66 pS (Zitt et al 1997;Hurst et al 1998;Kamouchi et al 1999) as well as a 17 pS subconductance (Kiselyov et al 1999) have been reported. Several studies suggest that TRPC3 channels display some constitutive activity (Hurst et al 1998;Dietrich et al 2003) and are activated in response to stimulation of cellular phospholipase C activity, involving the lipid mediator diacylglycerol but not depletion of intracellular Ca 2+ stores (Hofmann et al 1999;Lintschinger et al 2000;McKay et al 2000). However, another report demonstrates activation of this conductance in response to thapsigargin, a classical tool to deplete cellular Ca 2+ stores (Kiselyov et al 1999).…”