Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Abstract

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo-or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G q proteincoupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.Calcium signaling is involved in many cellular functions, including cell growth, differentiation, contraction, and secretion. The elevation of [Ca 2ϩ ] i occurs via two distinct phases upon activation of a G q protein-coupled receptor or a tyrosine kinase receptor. The first phase is mediated through the activation of phospholipase C, which catalyzes the production of inositol 1,4,5-trisphosphate, which in turn mobilizes Ca 2ϩ from the endoplasmic reticulum. The second phase of [Ca 2ϩ ] i elevation involves Ca 2ϩ entry from the extracellular milieu that maintains the [Ca 2ϩ ] i higher than the basal level (1, 2). The transient receptor potential canonical (TRPC) 3 protein family is involved in calcium entry and is composed of seven members (TRPC1 to TRPC7) that are calcium-permeable cation channels (3, 4). Based on sequence similarity, TRPCs can be further subdivided into four groups. Groups 1 and 2 contain exclusively the isoforms TRPC1 and TRPC2, respectively. Group 3 includes TRPC3, TRPC6, and TRPC7, whereas group 4 includes TRPC4 and TRPC5. Group 4 TRPCs are most closely related to group 1. TRPCs consist of a transmembrane domain composed of six segments with a putative pore between the fifth and the sixth transmembrane segments. The transmembrane domain is flanked with cytoplasmic N and C termini (5). TRPCs posse...

TheDrosophilaTRP and TRPL are assembled as homomultimeric channels in vivo

“…However, the TRP and TRPL do not heteromultimerize because of the difference in their C-termini, resulting in repulsion and dissociation of the subunits. This model fits well with previous in vitro results from Drosophila, as well as mammalian TRPC channels (Engelke et al, 2002;Lepage et al, 2006;Lepage et al, 2009;Liu et al, 2005;Xu et al, 2000;Xu et al, 1997), showing that N-terminal but not the C-terminal fragments of these channel subunits interact.…”

“…This model also explains why TRP and TRPL do not interact, as the C-terminal pattern of their cc-domains differs. However, this model is inconsistent with previous data showing that the N-terminal region determines subunit assembly, while the C-terminal region does not participate in subunit assembly (Engelke et al, 2002;Lepage et al, 2006;Lepage et al, 2009;Liu et al, 2005;Xu et al, 2000;Xu et al, 1997). In order to reconcile this apparent contradiction, we partially adapted a previously proposed model in which the N-terminal interaction assembles the channels.…”

“…Previous studies have demonstrated that coiled-coil (cc) domains and ankyrin repeats participate in TRP channel assembly (Engelke et al, 2002;Lepage et al, 2006;Lepage et al, 2009;Xu et al, 2000). In order to better understand previous (Xu et al, 1997) and current results and to explain the possibilities of association between TRP and TRPL, we analyzed the ankyrin repeats and cc-domains of TRP and TRPL channels (supplementary material Fig.…”

Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

“…Cardiac myocyte-targeted overexpression of either protein stimulates chamber hypertrophy and dysfunction coupled to Cn/NFAT activation in a feed-forward manner, because both channel genes also contain NFAT consensus sequences in their promoters (3,4). To date, genetic lossof-function studies have relied almost entirely on myocyte-targeted expression of dominant negative proteins (8) which suppresses hormone-stimulated or pressure-overload induced hypertrophy; however, because the channels can form heterotetramers (13), this approach may afford a poison peptide effect, in which a mutated channel protein interacts with other subtypes. If so, then this effect should differ from a pure gene deletion model, although this has not been reported to date.…”

TRPV4-mediated channelopathies

“…For example, in TRPC, tetramerization occurs through interaction of association domain 1 (AD1) (N-terminal region) followed by interaction with AD2 (putative pore region S4-S5 and C-terminal region). 13,51 However, in spite of several studies, the molecular mechanism underlying the assembly of TRPV monomers into functional tetramer is still at infancy. In addition, the regions of TRPV channels and the sequence specificity, which regulates the homo-or hetero-tetramer formation, are not well understood.…”