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We have tested the effects of decavanadate (DV), a compound known to interfere with ATP binding in ATP-dependent transport proteins, on TRPM4, a Ca2+-activated, voltage-dependent monovalent cation channel, whose activity is potently blocked by intracellular ATP4−. Application of micromolar Ca2+ concentrations to the cytoplasmic side of inside-out patches led to immediate current activation followed by rapid current decay, which can be explained by an at least 30-fold decreased apparent affinity for Ca2+. Subsequent application of DV (10 μm) strongly affected the voltage-dependent gating of the channel, resulting in large sustained currents over the voltage range between −180 and +140 mV. The effect of DV was half-maximal at a concentration of 1.9 μm. The Ca2+- and voltage-dependent gating of the channel was well described by a sequential kinetic scheme in which Ca2+ binding precedes voltage-dependent gating. The effects of DV could be explained by an action on the voltage-dependent closing step. Surprisingly, DV did not antagonize the effect of ATP4− on TRPM4, but caused a nearly 10-fold increase in the sensitivity of the ATP4− block. TRPM5, which is the most homologous channel to TRPM4, was not modulated by DV. The effect of DV was lost in a TRPM4 chimera in which the C-terminus was substituted with that of TRPM5. Deletion of a cluster in the C-terminus of TRPM4 containing positively charged amino acid residues with a high homology to part of the decavanadate binding site in SERCA pumps, completely abolished the DV effect but also accelerated desensitization. Deletion of a similar site in the N-terminus had no effects on DV responses. These results indicate that the C-terminus of TRPM4 is critically involved in mediating the DV effects. In conclusion, decavanadate modulates TRPM4, but not TRPM5, by inhibiting voltage-dependent closure of the channel.
TRPM4 is a Ca2+-activated but Ca2+-impermeable monovalent cation channel belonging to the melastatin subfamily of transient receptor potential (TRP) membrane proteins (Launay et al. 2002). Other unique properties of TRPM4, besides its activation by Ca2+, are its voltage dependence (Hofmann et al. 2003; Nilius et al. 2003) and block by ATP4− (Nilius et al. 2004). Ca2+-activated, non-selective cation channels (NSC) with properties reminiscent of TRPM4 and the homologous TRPM5 have been reported in various excitable and non-excitable cell types (Maruyama & Petersen, 1982; Suh et al. 1999, 2002; Koivisto et al. 2000; Ringer et al. 2000; Halonen & Nedergaard, 2002; Hurwitz et al. 2002; Magistretti & Alonso, 2002; Csanady & Adam-Vizi, 2003; Eto et al. 2003; Liman, 2003; Miyoshi et al. 2004; Rodighiero et al. 2004; Simard & Chen, 2004) exerting various cell functions, ranging from pacemaking and generation of cardiac afterdepolarizations (Guinamard et al. 2002, 2004), short-term memory (Egorov et al. 2002), vasomotor control (Suh et al. 2002) to volume regulation (Koch & Korbmacher, 1999) (for a review see Petersen, 2002). In particular, ATP-sensitive NSCs with an intrinsic voltage dependence and a single channel conductance of 25 pS comparable to that of TRPM4 have been observed in cardiomyocytes (Colquhoun et al. 1981; Guinamard et al. 2002, 2004; Wu, 2003; Zhainazarov, 2003.). Molecular identification of these TRPM4-like channels as well as understanding their function is hampered by the lack of selective modulating tools.
So far, TRPM4 channels have only been studied in heterologous expression systems (Launay et al. 2002; Nilius et al. 2003, 2004) but have also been identified as endogenous currents in HEK 293 cells (Launay et al. 2002). The functional analysis of this channel turned out to be relatively difficult because of the decay of channel activity in whole-cell and cell-free patch clamp measurements. The reason for this decay is not yet known. Very likely, the Ca2+ sensitivity of TRPM4 is regulated and partly or sometimes completely lost during the experiment, as has been suggested for the related TRPM5 desensitization (Liu & Liman, 2003).
This study focuses on properties of human TRPM4. In the first part, we present a quantitative approach to predict the observed changes in voltage dependence due to desensitization, indicating that Ca2+ and voltage sensitivity are interdependent. In the second part, we identify decavanadate (DV) as a strong modifier of TRPM4 channel gating. We were led by a recent report of Csanady & Adam-Vizi, 2004) who reported that the ATP block of Ca2+-activated non-selective cation channels in brain capillary endothelium is antagonized by DV, a compound known to interact with ATP binding sites. DV contains six negative charges that induce strong electrostatic interactions with sites accumulating positive charges, such as the ATP binding sites of various ABC ATPases, e.g. SERCA pumps (Toyoshima et al. 2000; Clausen et al. 2003) or the ATP, actin and DV binding myosin head segment, subfragment 1 (Tiago et al. 2004). Our results indicate that in contrast to the endogenous channels in brain capillary endothelium DV does not antagonize ATP block of TRPM4, but rather acts as channel activator by inhibiting voltage-dependent closure of the channel.
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The Ca2+-activated non-selective cation channel TRPM4 represents a molecular candidate for a large number of functionally similar Ca2+-activated cation channels found in native cells types (see Introduction). Typical fingerprints of this channel are its activation by Ca2+, its intrinsic voltage dependence and potent block by ATP4− (Launay et al. 2002; Nilius et al. 2003, 2004). We present here a sequential kinetic model in which Ca2+ binding precedes voltage-dependent gating, which is able to describe the observed Ca2+- and voltage-dependent gating behaviour of TRPM4 in inside-out patches. The analysis of channel activity in whole-cell experiments is hampered by a fast decay and mostly complete decay of the current (Nilius et al. 2003). Also in cell-free patches a fast current decay occurs immediately after patch excision, which levels off to a variable Ca2+-dependent rest activity. Measured from the same cells, the EC50 after current decay is much larger than the value immediately after patch excision, indicative of a decline of the Ca2+ affinity of the TRPM4 channel immediately after patch excision. The slower current decay at higher Ca2+ concentrations is compatible with this contention, because a much larger shift of the Ca2+ activation curve is required to significantly reduce the open probability. Interestingly, a similar desensitization of non-selective Ca2+-activated cation channels (NSC) has been observed in inside-out excised patches from pancreatic acinar cells (Maruyama & Petersen, 1984) and brain capillary endothelium (Csanady & Adam-Vizi, 2003). The rate of activation at positive potentials and the deactivation at negative potentials also declines during this desensitization phase, which is compatible with our model. The reason for desensitization is not yet understood, but it is likely that it will involve dephosphorylation processes or the loss of sensitizing cytosolic factors such as calmodulin. Ca2+ sensitivity of TRPM4 is very probably regulated. Desensitization might obviously also be the source for the scattering of the measured EC50 values for activation of TRPM4 by Ca2+ (compare also previously published values ranging from the submicromolar values to values exceeding 100 μm; Launay et al. 2002; Hofmann et al. 2003; Nilius et al. 2004). Even in our own studies, EC50 values measured in excised patches are scattered between 140 μm (this study) and 370 μm (Nilius et al. 2004). We describe here in detail that protocols for the measurement of the EC50 values for Ca2+ are hampered by desensitization (see supplementary data). Therefore, EC50 values for channels showing fast desensitization must be considered with caution. Some other reasons, which also apply for studies on TRPM5 showing the same kind of desensitization, can be understood from our quantitative approach. We describe here for the first time the interdependence of Ca2+ and voltage. The EC50 measurement results by definition from an estimation of PO/PO,max (see supplementary data). Therefore, the measured EC50 depends on both the ‘real’Kd value and on voltage. Changes in voltage dependence inevitably lead to changes in the EC50. As usual, measurements of EC50 values are performed after desensitization has reached a ‘steady state’. However, this steady-state level is variable even at the same Ca2+ concentration. A further reason for the scatter is an often overlooked methodological problem: Since it is very difficult to obtain the correct PO,max experimentally (in this case at high Ca2+ concentrations and very positive potentials), and the determination of EC50 strongly depends on the value of PO,max used for normalization, this can contribute to the scattering in inside-out experiments.
Our linear three-state kinetic scheme used for the description of dependence of PO and of τ of (de)activation of the TRPM4 currents on [Ca2+] and voltage during the stationary phase after patch excision was also used to characterize the effects of decavanadate, a compound that competes with ATP at ATP-binding sites, as described for ATP-dependent transport proteins, especially for SERCA Ca2+ pumps (Csermely et al. 1985; Hua et al. 2000; Toyoshima et al. 2000). It has also been described that DV increases single channel conductance of NSCs (Popp & Gögelein, 1992; Csanady & Adam-Vizi, 2003) and is an activator of NSC channels in endothelium by antagonizing the ATP block (Csanady & Adam-Vizi, 2004). Here we show that DV modulates channel activity of TRPM4 heterologously expressed in HEK 293 cells, mainly by interfering with channel gating. From our analysis we conclude that the most likely effect of DV is a dramatic shift of the voltage dependence of channel closing (β(V)) towards negative potentials, resulting in a strongly reduced voltage -dependence of PO in the investigated voltage range. As a consequence, robust inward currents occur at negative potentials, and the time-dependent components of the current during voltage step are small. This activation occurs in the micromolar range, and is not due to a substantial change in Ca2+ affinity of the channel. A similar effect of decavanadate has been recently described for NSCs in brain capillary endothelium (Csanady & Adam-Vizi, 2004), and was explained by a slowed channel closure caused by a high-affinity binding of DV to the open conformation of the channel (EC50 of 90 nm). In this same paper, Csanady & Adam-Vizi (2004) described an antagonistic effect of DV on the ATP block of these channels. They explained this ATP block by a high-affinity binding of ATP in the closed channel conformation, but competitively at the same site as DV. These effects of DV on ATP block are at variance with our present observation in TRPM4 channels, showing that DV actually sensitized ATP block and decreased the IC50 by a factor of 10. It is difficult to reconcile these data with a model whereby ATP and DV preferentially bind to the same site in the open and closed channel configuration, respectively. We assume that DV binds to a site, which interacts with the voltage-sensing mechanism (channel closing). The question remains how DV acts on TRPM4. Although it has been suggested that DV may act via lipid peroxidation of the membrane (Soares et al. 2003; Tiago et al. 2004), we think that this explanation is unlikely because of the very fast onset and the fast and complete reversibility of DV effects on TRPM4.
The unexpected finding that ATP blocks both inward and outward currents in the presence of DV suggests that the ATP block might be voltage independent, which is in contrast with our previous contention that ATP would act as an open pore blocker in the absence of DV. Our results therefore suggest that DV binds to a site, which modulates the voltage dependence of the channel rather than interfering with the blocking site of ATP4−.
To evaluate the mechanisms of DV action, we have first tested whether the closely related channel TRPM5 responds to DV. TRPM5 was insensitive to ATP4− at concentrations as high as 1 mm (N. D. Ullrich et al. unpublished observations). It might therefore not be completely unexpected that DV did not affect TRPM5. DV has been successfully used to identify the ATP binding site in SERCA (Toyoshima et al. 2000). Such a binding site shows some clear plasticity and depends on a structural motif rather than on a specific peptide sequence (Clausen et al. 2003). Decavanadate binding in the SERCA Ca2+ ATPase occurs in a spatial structure to which the nucleotide binding domain N, the actuator domain A, and the phosphorylation domain P all contribute (Hua et al. 2000). A putative ATP binding site in ABC ATPases is composed of elements with the sequences TETAL, FSRDRK, KGAPE, RCLALA (Clausen et al. 2003). Especially interesting are highly positively charged sites, which have also been identified in the head segment of myosin (called subfragment 1) and bind DV (Tiago et al. 2004). The intracellular domains of TRPM4 contain multiple regions with a high density of positively charged residues. One of these sites is located in the C-terminus of TRPM4 and confers a stretch of six amino acids with four positive and one negative charge (136RARDKR, R/K mutant). Such a motif is lacking in TRPM5. We have therefore first constructed a chimera of TRPM4 containing the C-terminus of TRPM5. This chimera shows some properties of TRPM5, i.e. a complete and rapid desensitization after patch excision, but lacks any effect of DV. Likewise, the R/K mutant also showed a complete lack of DV effect. Interestingly, the R/K mutant and also the chimeras showed changes in desensitization. It can be speculated that these sites are important for regulation of the Ca2+ sensitivity of TRPM4 and may be also involved in ATP binding. However, such a possible binding is different from the blocking site, because the chimeric channels showed a similar block by ATP4− as the wild type TRPM4 channels (IC50= 0.5 μm ATP4−, data not shown, n= 3 for three concentrations).
A similar motif was found in the N-terminus of TRPM4, namely 332RDRIRR, which also comprises four positive charges and one negative charge. Deletions of these motifs resulted in functional channels, which could still be, in contrast to the C-terminal deletion, modulated by DV. All these data together suggest that the C-terminus of TRPM4 is crucially involved in regulation of gating, and is at least part of the DV acceptor that can dramatically modulate the kinetic behaviour of this channel. However, this C-terminal site is very probably different from the blocking site for ATP4−.
In conclusion, we have identified DV as the first strong modulator of voltage-dependent gating in the Ca2+-activated cation channel TRPM4. DV might represent a novel tool to modulate endogenous TRPM4-mediated NSCs and may provide a possible way to differentiate between TRPM4 and TRPM5, and may contribute to our understanding of TRPM4 gating. Finally, it is tempting to speculate that an endogenous molecule with properties similar to those of DV may act as a physiological ligand for TRPM4.