Canonical transient receptor potential (TRPC) 1 acts as a negative regulator for vanilloid TRPV6-mediated Ca2+ influx.

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca(2+) selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca(2+) and Na(+) currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca(2+) influx in HEK293 cells.

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels.

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Förster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.

Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation.

The Ca(2+)-selective TRPV6 as well as the L-type Ca(2+) channel are regulated by the Ca(2+)-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca(2+), employing Ca(2+)-imaging and patch-clamp techniques. Additionally, confocal Förster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein-protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca(2+)-insensitive mutant (CaM(MUT)) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca(2+)/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6-CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca(2+)-insensitive CaM(MUT), however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaM(MUT) with rTRPV6, suggesting a strict Ca(2+) dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca(2+) levels, as observed by dynamic FRET measurements. An rTRPV6Delta(695-727) mutant with the very C-terminal end deleted, yielded Ca(2+) currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca(2+) channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695-727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.

TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Voltage-gated rearrangements associated with differential beta-subunit modulation of the L-type Ca(2+) channel inactivation.

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.

CaT1 knock-down strategies fail to affect CRAC channels in mucosal-type mast cells.

CaT1, the calcium transport protein 1 encoded by TRPV6, is able to generate a Ca(2+) conductance similar but not identical to the classical CRAC current in mucosal-type mast cells. Here we show that CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited either by expression of various dominant negative N-terminal fragments of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense suppression. By contrast, the endogenous CRAC current of the mast cells was unaffected by CaT1 antisense and siRNA knockdown but markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant negative N-CaT1 fragments. Inhibition of CRAC current was not an unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents of the mast cells were not significantly affected by these N-CaT1 fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived currents in mast cells, but failed to inhibit CRAC currents. Thus, the structural requirements of rCaT N-terminal fragments for inhibition of rCaT1 and CRAC channels are different. These results together with the lack of CaT1 antisense and siRNA effects on currents render it unlikely that CaT1 is a component of native CRAC channels in mast cells. The data further demonstrate a novel strategy for CRAC current inhibition by an N-terminal structure of CaT1.

Oriented binding of the His6-tagged carboxyl-tail of the L-type Ca2+ channel alpha1-subunit to a new NTA-functionalized self-assembled monolayer.

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His6-tagged carboxyl-tail (C-tail) of the alpha1c-subunit of the L-type Ca2+ channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the His6-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice. The topography of this surface was characterized using AFM; its ability to bind C-tail proteins oriented and stable was confirmed by SPR measurements and by complementary force spectroscopy experiments with a CaM4-construct covalently attached to the tip. The developed anchoring strategy can now be used to study receptor/ligand interactions in general applying force spectroscopy and SPR on His6-tagged proteins oriented immobilized onto this new NTA-functionalized self-assembled monolayer.

Adhesion and proliferation of human vascular cells on UV-light-modified polymers.

We studied the adhesion and proliferation of human umbilical-vein endothelial cells (HUVEC) and human aortic smooth-muscle cells (HASMC) on modified polymer samples. The polymers under investigation were either PTFE (polytetrafluoroethylene) or PET [poly(ethylene terephthalate)], which are among the most frequently employed biomaterials in reconstructive medicine. The polymer surfaces were modified by exposure to the UV light of an excited-xenon-dimer (Xe2*) Excimer lamp (Heraeus-Noblelight, Hanau, Germany) at a wavelength of 172 nm in an NH3 atmosphere. On modified PTFE, this resulted in a significant increase in the number of adhering HUVEC or HASMC 1 day after seeding and in the formation of a confluent cell layer after 3-4 days. On PET, HUVEC adhesion and proliferation rates were already high on untreated samples and could not be significantly increased by the UV-light treatment, but the cells were distributed more homogenously on the treated samples. Cell proliferation was in all cases comparable with, or even better than, that obtained on standard polystyrene (PS) Petri dishes used in cell cultivation. As on PS Petri dishes, the proliferation of HASMC on modified PTFE was faster than that of HUVEC under similar culture conditions.

Store depletion-activated CaT1 currents in rat basophilic leukemia mast cells are inhibited by 2-aminoethoxydiphenyl borate. Evidence for a regulatory component that controls activation of both CaT1 and CRAC (Ca(2+) release-activated Ca(2+) channel) channels.

The intestinal Ca(2+) transport protein CaT1 encoded by TRPV6 has been reported (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709) to be all or a part of the Ca(2+) release-activated Ca(2+) channel (CRAC). The major characteristic of CRAC is its activation following store depletion. We expressed CaT1 in HEK293 cells and rat basophilic leukemia (RBL) mast cells and measured whole-cell currents by the patch clamp technique. In HEK293 cells, the expression of CaT1 consistently yielded a constitutively active current, the size of which was strongly dependent on the holding potential and duration of voltage ramps. In CaT1-expressing RBL cells, the current was either activated by store depletion or was constitutively active at a higher current density. CaT1 currents could be clearly distinguished from endogenous CRAC by their typical current-voltage relationship in divalent free solution. 2-aminoethoxydiphenyl borate (2-APB), which is considered a blocker of CRAC, was tested for its inhibitory effect on both cell types expressing CaT1. Endogenous CRAC as well as store-dependent CaT1-derived currents of RBL cells were largely blocked by 75 microm 2-APB, whereas constitutively active CaT1 currents in both RBL and HEK293 cells were slightly potentiated. These results indicate that despite the difference in the permeation properties of CRAC and CaT1 channels, the latter are similarly able to form store depletion-activated conductances in RBL mast cells that are inhibited by 2-APB.