Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Cation channels which allow permeation of Na⁺ as well as K⁺, and to some extent also permeation of divalent cations, are present in in the plasma membrane of endothelial cells [1-5]. Endothelial non-selective cation channels are activated in response to stimulation of phospholipase C-coupled receptors or depletion of Ca²⁺ stores [1-3] and during oxidative stress [4,5]. The molecular nature and the exact role of these channels is still elusive. One family of membrane proteins that has been reported to constitute cation channels of variable ion selectivity is the Trp (Drosophila transient receptor potential gene product) protein family [6]. It appears likely that a variety of TRP channels with different activation and permeation properties are formed by heteromultimerization of Trp proteins. Recently, evidence has been presented for the expression of Trp homologues in vascular endothelium [7,8]. Here we present evidence that Trp homologues form non-selective cation channels of physiologic and pathophysiologic relevance in vascular endothelial cells.

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Fig. 1: Expression of different Trp isoforms in HUVEC.
Trp genes (Trp 1, 3 and 4) were amplified by RT-PCR, blotted and probed with digoxygenin-labeled oligonucleotides internal to the PCR primers used. Two different sets of primers were used to amplify Trp4: one was to bind to mTrp4 (indicated as Trp4), the other one to hTrp4 (indicated as *Trp4). RT-PCR experiments were performed with total RNA from HUVEC and HEK293. Total RNA from HUVEC was subjected to PCR without preceeding reverse transcription as a negative control. Three available cDNA clones (hTrp1, hTrp3 and mTrp4) were used as PCR templates for a positive control.

The results indicate expression of three human Trp species in human umbilical vein endothelium. Similarily, RT-PCR products of the expected size (448 bp) were obtained from total RNA preparations of ECAP with primers specific for hTrp3, indicating the expression of a Trp3 isoform in porcine aortic endothelial cells (not shown).

Depletion of intracellular Ca²⁺ stores and oxidative stress are associated with activation of a non-selective cation conductance

Depletion of intracellular Ca²⁺ stores by dialyses of HUVEC with IP₃ plus the Ca²⁺ chelator BAPTA (10 mM) induced a substantial increase in membrane conductance. The increase in inward current at -80 mV was larger and more stable with Na⁺ (137 mM) than with Ca²⁺ (10 mM) as charge carrier (not shown). Typical membrane currents recorded in a Na⁺ -containing, Mg²⁺ -free solution are shown in Fig. 2.

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Fig. 2: Ca²⁺ store depletion by IP₃ induces a cation current in HUVEC.
HUVEC were dialyzed with IP₃ (100 µM) via the pipette solution starting with rupture of the patch at time 0. Upper panel: Time-course of membrane current at 80 mV is shown for a cell which was transiently transfected with GFP. Removal of extracellular Na⁺ and addition of La³⁺ (50 µM) is indicated. Lower panel: Current to voltage relationship derived at the time points indicated.

Fig. 2. shows the time course of the membrane current recorded at -80 mV (upper panel). This current was dependent on extracellular Na⁺ blocked by 50µ M La³⁺ (N = 7). Lower panel of Fig. 2 illustrates the current to voltage relationship of the IP₃ -induced current that reversed at about neutral potential. Removal of extracellular Na⁺ resulted in a reduction of current and shifted the reversal potential in hyperpolarizing direction indicating that the current is carried for a large part by Na⁺. The IP₃ -induced cation conductance was observed with high reproducibility (14 out of 15 cells).
ECAP exhibited very small IP₃ -induced membrane currents. However, a current reminiscent of IP₃ -induced membrane currents was observed after prolonged exposure (60 min) of cells to the lipophilic oxidant tBHP (400 µ M) Oxidative stress resulted in a dramatic increase in membrane conductance of ECAP. As shown in Fig. 3., large inward currents were detectable in tBHP-treated ECAP at negative membrane potentials (-80 mV).

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Fig. 3: tBHP activates a cation conductance in ECAP.
Upper panel: Time course of membrane currents at -80 mV recorded from a cell after exposure to tBHP (400 µM, 60 min). Removal of extracellular Na⁺ [Na⁺e] leads to reversible shift in the current to voltage relationship. Lower panel: current to voltage relationships derived at the time points indicated in the upper panel.

Removal of extracellular Na⁺ reduced the inward currents at -80 mV due to a shift of the zero potential to more negative values indicating a significant contribution of Na⁺ to the tBHP-induced current. Similarily to the IP₃-induced current of HUVEC, the oxidant-induced current was completely blocked by 50 µM La³⁺ (data not shown).

Endothelial non-selective membrane conductances are eliminated by expression of the N-terminal domain of hTrp3

The N-terminal domain (residues 1-302) of hTrp3 was cloned into the vector pIRES-EGFP which allows for simultaneous expression of proteins and the marker protein GFP. Endothelial cells were transiently transfected with N-TRP in pIRES-EGFP. The rational for this strategy is the observation that N-terminal fragments of Trp proteins exert a dominant negative effect on Trp channel function [13]. Control experiments were performed in cells transfected pIRES-EGFP only. Some experiments were performed with cells transfected with full length hTrp3 which is expected to interact with endogenous Trp proteins without preventing the formation of functional Trp oligomers. Alternatively, cells were transfected with C-TRP (residues 721-848 of hTrp3). HUVEC transfected to express GFP only, responded to intracellular administration of IP₃ with a clear increase in the membrane current recorded at -80 mV (N=14). As illustrated in Fig. 4A, the inward current recorded at -80 mV in N-TRP-transfected HUVEC remained stable during dialysis with IP₃ (100 µ M; 10 - 20 min recording time; N = 15). Cells transfected with hTrp3 responded to intracellular administration of IP₃ in a manner similar to GFP-transfected controls, exhibiting even slightly larger increases in membrane conductance.

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Fig. 4: Modification of endothelial cation currents by expression of TRP proteins.
A. Mean inward currents (at 80 mV), recorded initially (open columns) and 4-6 min after obtaining the whole cell configuration with IP₃-containing pipette solution (filled columns) in cells transfected with GFP only (control/GFP; N=14), or NTRP3 plus GFP by transfection of NTRP3 in pcDNA3-pIRES-EGFP (NTRP3/GFP*; N=5) or hTrp3 plus GFP (hTrp3/GFP; N=5).
B. Expression of NTRP3 but not CTRP3 suppresses tBHP-induced cation conductance. Membrane currents at -80 mV in sham-transfected endothelial cells without (-tBHP; N=4) or with pretreatment of cells with the oxidant (+tBHP; N=12), NTRP3-transfected (+tBHP; N=14) and CTRP3-transfected (+tBHP; N=5).
Mean values S.E.M.;* indicates significant differences versus initial current value (A) or versus control + tBHP (B) respectively.

Transfection of HUVEC with N-TRP clearly suppressed the IP₃/store depletion-induced cation conductance. It is of note that other characteristic membrane conductances such as the inwardly rectifying K⁺ conductance appeared unaffected (not shown).
To test whether Trp proteins are involved in the tBHP-induced cation conductance, ECAP were transiently transfected to express two different cytosolic domains of hTrp3: either i) the amino terminal domain (amino acid residues 1-302, referred to as N-TRP), or ii) the carboxy terminal domain (amino acid residues 721-848, referred to as C-TRP). The transfection procedure itself did not affect the response of ECAP to tBHP (sham transfection) as shown in Fig. 4B. Similarly, ECAP expressing only GFP due to transfection with pIRES-EGFP displayed no modification of the response to tBHP (not shown). In clear contrast, transfection of ECAP with the N-TRP construct resulted in a significant suppression of the tBHP-induced current at -80 mV. By contrast, transfection of ECAP with C-TRP failed to suppress the inward currents induced by tBHP.

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In summary, our results suggest that homologues of the Drosophila transient receptor potential (Trp) gene product contribute to two distinct non-selective cation conductance of vascular endothelial cells, i.e. an IP₃ -induced cation conductance which may serve physiologic activation of endothelial cells and a rather pathophysiologically relevant cation conductance which is activated by oxidative stress. Our conclusion is based on the observation that expression of a N-terminal fragment of a Trp protein (N-TRP) that is known to exert a dominant negative effect on Trp channel function, eliminates both cation conductances. The well documented ability of N-TRP to bind to the N-terminal domain of other Trp proteins [13] is expected to result in an interaction of N-TRP with endogenous Trp proteins and consequently in suppression of the assembly of functional Trp oligomers. Our results favour the idea of an involvement of Trp proteins in the phenomena of IP₃ - and oxidant-induced cation conductances of endothelial cells. Expression of complete hTrp3 or of a C-terminal fragment of hTrp3 (C-TRP) failed to affect non-selective conductances in endothelial cells. Expressed hTrp3 is expected to associate with endogenous Trps without essential impairment of channel function and expression of a C-terminal fragment is not expected to interfere with assembly of endogenous Trp channels. Despite the apparent differences in the mechanism of activation, IP₃- and oxidant-induced cation conductances of endothelial cells exhibited strikingly similar properties, i.e. sensitivity to inhibition by N-TRP, a rather high permeability for Na⁺ ions and relatively high sensitivity to block by La³⁺. Activation of cation channels by intracellular administration of IP₃ in the presence of the Ca²⁺ chelator BAPTA may involve either a direct interaction of IP₃ with the channel or a mechanism related to depletion of intracellular Ca²⁺ stores. In particular the latter mechanism has been implicated in activation of specific isoforms of Trp [6]. Our present knowledge on Trp proteins suggest an important role of some species in hormone-regulated Ca²⁺ but also of monovalent cation conductances [6,14,15]. Expression of some members of the Trp protein family such as Trp1 and 3 gave rise to poorly selective cation conductances which allow for large Na⁺ currents [15] similar to that observed in endothelial cells upon exposure to oxidative stress. Oxidative stress by itself does not release Ca²⁺ from intracellular stores [16]. Thus, it appears unlikely that oxidative stress activates Trp channels by depletion of Ca²⁺ stores.On the other hand, direct redox sensitivity of Trp channels has, to our knowledge, not yet been studied. Nonetheless, a mechanism of activation independent of intracellular Ca²⁺ handling such as oxidation of critical sulfhydryl groups due to accumulation of oxidized glutathione [4,5] (see below Fig.5) may well be considered for Trp channels. Non-selective Trp channels may, to some extent, contribute to physiologic Ca²⁺ homeostasis. Excessive activation of such Trp channels, however, is expected to result in cellular Na⁺ loading and membrane depolarization as observed in oxidative stress. Membrane depolarization is known to suppress Ca⁺ signalling in endothelial cells. The consequences of Na⁺ loading on the other hand are so far barely understood. Changes in the intracellular Na⁺ concentration may well interfere with cellular Ca²⁺ signals and regulation of endothelial functions due to control of subcellular Ca⁺ gradients via Na⁺/Ca²⁺ exchange [17].
So far up to five trp genes were detected in vascular endothelial cells [4; 5]. Formation of various heteromultimeric Trp complexes [14] may well give rise to a functional diversity among the Trp channels in specific tissues. Thus, it appears conceivable to speculate that Trp heteromultimers are the molecular basis more than one cation conductance in endothelial cells. One group may serve hormonal control of Ca²⁺ homeostasis while another group may play a role in pathophysiologic situations such as oxidative stress as (Fig. 5).

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Fig. 5: Hypothetical functions of endothelial TRPs.
TRP proteins may be involved in multiple ion conductances of endothelial cells. These TRP-mediated conductances include a store-operated Ca²⁺ entry pathway and a redox-activated Na⁺ conductance. Abbreviations used: ER: endoplasmatic reticulum; PLC: phospholipase C; PIP₂: phosphatidylinositolbisphosphate; IP₃: inositoltrisphosphat; GSH: glutathione; GSSG: oxidized glutathione

In summary, we suggest a central physiologic as well as pathophysiologic role of non-selective cation channels formed by Trp heteromultimers in endothelial cells. Endothelial Trp proteins may on the one hand serve hormonal control of Na⁺ and Ca²⁺ homeostasis and in addition determine cellular redox sensitivity.

The authors wish to thank Dr. M. Poteser for his efforts preparing this manuscript and Dr. X. Zhu for kindly providing TRP cDNA-clones.
The work was supported by the Austrian Research Funds, SFB Biomembranes F708 and F715 as well as P12667.

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