| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Experimental Cardiac Surgery, Heart and Vessel Diseases, 2Laboratory of Experimental Cardiology, and 3Laboratory of Physiology, Katholieke Universiteit Leuven, Leuven, Belgium
Submitted 16 February 2006 ; accepted in final form 10 May 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells IMIC exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(
,
-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented IMIC rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 µM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by prevention of its resynthesis (10 µM wortmannin or 15 µM phenylarsine oxide) induced rundown of IMIC. Finally, loading ATP-depleted cells with exogenous PIP2 (10 µM) prevented rundown. These results suggest that PIP2, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity. cation channels; hydrolysis; phosphoinositides; rundown
We previously described (10, 31, 48) a Mg2+-inhibited cation (MIC) channel in cardiac myocytes that displays pharmacological and pore properties resembling those of TRPM6 and TRPM7 channels. Native TRPM7-like currents such as cardiac MIC are also referred to as magnesium-nucleotide-regulated metal ion (MagNuM) currents (32). TRPM6 and TRPM7 are closely related members of the TRPM subfamily, which form channels with very similar properties (29, 45). TRPM6 is mainly expressed in the kidney and small intestines, and its mutation causes hypomagnesemia with secondary hypocalcemia (5, 41, 46). TRPM7 is widely expressed with high levels in the kidney and heart. It is essential for cell viability and proliferation and is also involved in the homeostasis of Mg2+ and probably other metals in various tissues (11, 14, 19, 32, 39, 42), whereas in cortical neurons it is implicated in anoxic cell death (1).
The regulation of TRPM6 and TRPM7 channels is incompletely understood. Both TRPM6 and TRPM7 channels contain functional kinase domains and are regulated by cytosolic levels of Mg2+. TRPM7 channels have been proposed to be regulated by MgATP, MgGTP, ATP, cAMP, or phosphatidylinositol 4,5-bisphosphate (PIP2) (reviewed in Refs. 8, 34). Like TRPM6 and TRPM7 channels, cardiac and smooth muscle MIC channels are mainly regulated by cytosolic Mg2+ (48), but the slow increase of whole cell MIC current (IMIC) on cell dialysis with low-Mg2+ solution suggests that other factors apart from a simple washout of intracellular Mg2+ (35) are involved. We observed (10) that in ATP-depleted cells IMIC was not sustained but decreased with time until complete loss. Rundown of IMIC has been reported previously (21), but a link with ATP has not been established. Therefore, we investigated the role of intracellular ATP in the regulation of cardiac MIC channels and show that PIP2, probably generated by ATP-utilizing lipid kinases, is necessary for maintaining the activity of the channels.
A preliminary report of these findings has been published in abstract form (9).
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
We used cardiac ventricular myocytes isolated mostly from pig hearts but also from guinea pig and rat. The methods for cell dissociation and electrophysiological measurements were similar to those described in detail previously (26). Briefly, pigs were premedicated with azaperone (4 mg/kg im) and atropine (0.35 mg/kg iv). They were then anesthetized with pentobarbital sodium (5–15 mg/kg iv), intubated, and ventilated with 40% oxygen in air, and then injected with heparin (5–8 mg/kg iv) and a lethal dose of pentobarbital sodium (100 mg/kg) before thoracotomy and tissue extraction. Unpremedicated guinea pigs and rats were injected with heparin (70–85 mg/kg ip) 10 min before injection with pentobarbital sodium (150–300 mg/kg ip). The cell dissociation procedure consisted essentially of an enzymatic tissue digestion during Langendorff perfusion with oxygenated Ca2+-free Tyrode solution at 37°C. After the washout of enzymes, Ca2+ was gradually reintroduced up to a concentration of 0.18 mM. The tissue was cut into chunks, and cells were dissociated by gentle mechanical agitation and filtered through a mesh (200-µm hole diameter). Cells were allowed to settle down at room temperature in 0.18 mM Ca2+ Tyrode solution, and those not immediately used were stored at 4°C.
Whole cell currents were recorded with the patch-clamp technique at room temperature. Patch pipettes were pulled from capillary glass (GB 200-8P; Science Products, Hofheim, Germany) on a DMZ-Universal Puller (Zeitz-Instrumente, Munich, Germany), which fire-polished the electrodes to a final resistance of 2–4 M
when filled with internal solution. To measure TRPM7-like currents the voltage-clamp protocol consisted of 4-s symmetrical voltage ramp commands from –120 mV to +80 mV and back to –120 mV, applied every 10 s from a holding potential of –80 mV. The slow rate of depolarization (0.1 V/s) during the ascending limb allowed for complete inactivation of the voltage-dependent Na+ and T-type Ca2+ currents, whereas the L-type Ca2+ channels were blocked by nifedipine (100 µM) included in all extracellular solutions. Currents were measured during the descending limb of the voltage ramp. In a few cells, the L-type Ca2+ current was measured by initially stepping from the holding potential to –40 mV for 0.6 s to inactivate voltage-dependent Na+ current; the Ca2+ current was then induced by depolarizing to 0 mV for 0.2 s. Nifedipine was not included in the extracellular solution in these experiments. Voltage protocols were generated and data recorded with the pCLAMP 8.1 software via a Digidata 1322A acquisition system (Axon instruments, Union City, CA). Data were filtered at 1 kHz and sampled at 5 kHz. A 10-mV voltage pulse from the holding potential was used to measure cell capacitance (122 ± 5 pF; n = 59), which remained stable during measurements even after completion of the rundown process described below (in 13 cells, 118 ± 11 pF on patch rupture, 120 ± 8 pF after rundown).
Solutions and drugs.
The standard Tyrode solution used for tissue perfusion during cell isolation contained (mM) 135 NaCl, 5.4 KCl, 0.9 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose; pH was adjusted to 7.4 with NaOH. During measurements, the myocytes were superfused with a solution of similar composition except that K+ was replaced by Cs+. Nominally Ca2+-free or Mg2+-free solutions were prepared by simply omitting these ions from the standard solution. The standard pipette solution contained (in mM) 130 Cs-glutamate, 25 CsCl, 1 MgCl2, 5 Na2ATP, 1 EGTA, 0.1 Na2GTP, and 5 HEPES (pH 7.25; adjusted with CsOH). The pipette solution was modified by changing levels of Mg2+ or ATP, by substituting ATP with its nonhydrolyzable analog adenosine 5'-(,-imido)triphosphate (AMP-PNP), or by adding specific drugs when necessary. Free internal Mg2+ and ATP concentrations were calculated with CaBuf software (courtesy of Prof. G. Droogmans, Laboratory of Physiology, University of Leuven).
Genistein, daidzein, forskolin, and okadaic acid were obtained from Tocris Cookson (Bristol, UK), whereas KT-5720, chelerythrine, cyclosporin A, wortmannin, and U-73122 were from Alomone Labs (Jerusalem, Israel). D-myo-phosphatidylinositol 4,5-bisphosphate (PIP2) was obtained from Echelon Biosciences (Salt Lake City, UT), and PIP2 antibody was from Gentaur Molecular Products (Brussels, Belgium). Solutions containing PIP2 or its antibody were sonicated before use. In initial experiments, we experienced difficulties with gigaohm seal formation with pipettes containing the PIP2 antibody. Therefore, we subsequently filled the pipette tip with plain internal solution before adding the antibody in the pipette shank. All other drugs or chemicals were from Sigma-Aldrich (Bornem, Belgium) or Merck (Darmstadt, Germany). Nifedipine (100 µM), made from stock solution (50 mM) prepared in ethanol, was used while protected from light in extracellular solutions to block L-type Ca2+ channels. KT-5720, genistein, daidzein, cyclosporin A, okadaic acid, wortmannin, phenylarsine oxide (PAO), and U-73122 were prepared as stock solution in dimethyl sulfoxide (DMSO) and diluted to the desired final concentrations. Levels of DMSO up to 1% that we tested on MIC channels had no effect.
Data and statistical analyses.
Data were analyzed with Clampfit 8.1 (Axon Instruments) and Origin 7 (Microcal, Northampton, MA). Average data are expressed as means ± SE, with n indicating the number of cells studied under each condition. Means were compared with the two-tailed Student's t-test, whereas differences among multiple groups were evaluated with ANOVA. P 
0.05 was taken as the threshold for statistical significance.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
20 µM), as well as the effect of removing extracellular divalent cations and the influence of internal ATP on the time evolution of the unmasked IMIC. In Fig. 1, A and C, traces of whole cell currents obtained in the presence of extracellular divalent cations immediately after patch rupture, at maximum amplitude, and at 50 min after patch rupture are superimposed. Cells were dialyzed with either ATP-containing (Fig. 1A) or ATP-free (Fig. 1C) internal solutions. Under such conditions, the effect of cell dialysis with low internal free Mg2+ typically consisted of a large increase of outward current and little or no change of inward current. The outward current remained stable in ATP-containing cells but decreased with time in ATP-depleted cells. In Fig. 1, B and D, extracellular divalent cations were removed after an initial control recording in the presence of extracellular Ca2+ and Mg2+. The other traces were taken at maximum amplitude and at 50 min after patch rupture. In addition to the increase of outward current largely due to the low internal Mg2+, large inward currents were induced because of a removal of IMIC block by extracellular Ca2+ and Mg2+ (IMIC reversal potential: –2.3 ± 1.0 mV; n = 35). In this case, too, IMIC was stable after reaching its maximum value in ATP-loaded cells (Fig. 1B) but decreased in ATP-depleted cells (Fig. 1D).
|
|
|
|
|
Requirement of ATP hydrolysis during MIC channel activity.
Because under physiological conditions free ATP levels are relatively low, we tested for the effects of low ATP on the time course of IMIC by dialyzing cells with internal solution containing 0.5 mM ATP (free ATP 0.26 mM, free Mg2+ 20 µM) instead of 5 mM ATP (free ATP 3 mM). Under such conditions, IMIC could be induced and was sustained both in the presence and in the absence of external divalent cations (Fig. 3, A and B).
|
Effects of protein kinase inhibitors on MIC channels. Because ATP acts as substrate in protein kinase-mediated phosphorylation that may regulate ion channels (15), we examined the involvement of classic protein kinases in the modulation of MIC channel activity. Figure 4 shows the effects of protein kinase inhibitors, applied in divalent cation-free external solutions, on IMIC in cells dialyzed with ATP-containing solution. The protein kinase A (PKA) inhibitor KT-5720 (1 µM; Fig. 4A), the protein kinase C (PKC) inhibitor chelerythrine (2.5 µM; Fig. 4B), and the broad-spectrum protein kinase inhibitor staurosporine (1 µM) had no effect on IMIC. Figure 4C shows that the protein tyrosine kinase inhibitor genistein (50 µM), but not its inactive analog daidzein (50 µM), partially and reversibly inhibited IMIC, consistent with results obtained in brain microglia (18). Data from different cells (n = 4–8 for each drug) confirm that none of the drugs except genistein caused any significant change of IMIC (Fig. 4D; P = 0.36 for all drugs except genistein, ANOVA; P < 0.01 for genistein vs. control, t-test). A similar lack of effect with KT-5720 or chelerythrine and a decrease of outward current with genistein were observed in the presence of external divalent cations (n = 2 for each drug).
|
-adrenergic receptor activation by isoproterenol (3 µM) does not affect IMIC. To verify that the adenylate cyclase-PKA cascade was not inactivated under our experimental conditions, we tested whether forskolin could modulate the L-type Ca2+ current (ICaL) in four cells dialyzed with our standard, ATP-containing solutions. Forskolin (10 µM) enhanced ICaL to 315 ± 15% of control (P < 0.01 vs. control; not illustrated). Altogether, the results suggest that among the classic protein kinases protein tyrosine kinase, but not PKA or PKC, modulates the activity of cardiac MIC channels. Effects of protein and lipid phosphatase inhibitors on MIC channels. Next, we tested for the sensitivity of the rundown process to protein phosphatase inhibitors. We used the protein phosphatase 1 and 2A inhibitor okadaic acid (12), the protein phosphatase 2B inhibitor cyclosporin A (37), and the protein tyrosine phosphatase inhibitor sodium orthovanadate on cells dialyzed with ATP-free solution. A combination of okadaic acid (10 µM) plus cyclosporin A (10 µM) was given internally via cell dialysis, whereas sodium orthovanadate (1 mM) was applied externally. As shown in Fig. 6D, after 60 min of cell dialysis in the presence of these drugs, IMIC had decreased to <40% of its maximum value, which was not different from the decrease in control ATP-depleted cells (P = 0.29 for okadaic acid + cyclosporin A vs. control; P = 0.27 for orthovanadate; n = 4 cells in each case).
Because of the failure of protein phosphatase inhibitors to prevent rundown of IMIC, we tested for the effects of lipid phosphatase inhibitors. We used a cocktail of phosphatase inhibitors, fluoride + vanadate + pyrophosphate (FVPP; F– 5 mM; VO
0.1 mM; pyrophosphate 10 mM) (17), applied internally in ATP-depleted cells. With FVPP, IMIC was sustained in the presence (Fig. 6A) as well as in the absence (Fig. 6B) of external divalent cations. After 60 min of cell dialysis IMIC was 93 ± 2% of its maximum level (Fig. 6D; P < 0.01 relative to control, n = 4), suggesting that FVPP could prevent rundown. Thus the putative dephosphorylation process mediating rundown of IMIC seems to involve lipid, but not protein, phosphatases. As shown above, tyrosine kinase modulates MIC channels, but the phosphorylation mediated by this enzyme does not appear to be the main process preventing IMIC rundown.
Dependence of MIC current on PIP2.
The modulation by lipid phosphatases and the need for ATP hydrolysis suggest the involvement of lipid kinases in the maintenance of MIC channel activity. ATP acts as substrate for lipid kinases that phosphorylate phosphatidylinositol (PI) to higher-order phosphoinositides. Among these phosphoinositides, PIP2 modulates many ion channels (7, 16, 43). Its precursor, phosphatidylinositol 4-phosphate, is synthesized from PI by a reaction catalyzed by phosphatidylinositol 4-kinase (PI4-kinase) that can be inhibited by wortmannin at high concentrations (IC50 50–140 nM; Refs. 28, 33) or by PAO (47). Because the MIC channels resemble TRPM7 channels, which are regulated by PIP2 (38), we hypothesized that the ATP requirement of cardiac MIC channels may be related to PIP2 metabolism. Figure 7A shows the effect of PIP2 depletion when its resynthesis is prevented by internal application of wortmannin (10 µM) in ATP-loaded cells. Under these conditions IMIC underwent rundown, decreasing to 37 ± 8% at 50 min after patch rupture (n = 4, P < 0.01 vs. control; Fig. 7D). Rundown was also induced in guinea pig ventricular myocytes by wortmannin (15 µM) applied externally (IMIC at 50 min after patch rupture: 21 ± 12% of maximum; n = 4). Because phosphatidylinositol 3-kinase (PI3-kinase) is also (and more potently) inhibited by wortmannin (IC50 = 3–5 nM; Ref. 33) we sought to exclude its involvement. We dialyzed ATP-loaded cells with 300 nM wortmannin and measured IMIC in the presence of external divalent cations. Under these conditions there was no rundown (at 50 min after patch rupture relative IMIC = 92 ± 3%, n = 3, P = 0.41 vs. control untreated cells; not illustrated), suggesting that rundown was not due to PI3-kinase inhibition. In addition, we used PAO as an alternative PI4-kinase inhibitor on ATP-loaded cells. Figure 7, B and D, illustrate that PAO (15 µM) induced a slow decrease of IMIC to 50 ± 8% of predrug level after 20 min of drug application (P < 0.01 vs. predrug level, n = 6). These results suggest that loss of PIP2 may underlie reduction of IMIC.
|
To test the effects of PIP2 more directly, we examined the time course of IMIC in ATP-depleted cells dialyzed with exogenous PIP2 (10 µM). Under these conditions, IMIC in the presence (Fig. 8A) or absence (Fig. 8, B and C) of external divalent cations was sustained, although fluctuations in the amplitude of monovalent inward currents were observed in some cells (Fig. 8C). Pooled data of outward currents at +80 mV in the absence of external divalent cations are shown in Fig. 8D and confirm that exogenous PIP2 could prevent rundown in ATP-depleted cells (at 50 min, relative IMIC = 98 ± 2%, n = 7; P < 0.01 vs. control without PIP2). There was no significant difference between maximal IMIC measured in ATP-depleted cells dialyzed with (9.2 ± 1.2 pA/pF, n = 7) or without (10.4 ± 1.0 pA/pF, n = 10) PIP2. The shape of the current-voltage relationships and the reversal potential of monovalent cation currents (–1.9 ± 2.9 mV, n = 7) during dialysis with PIP2 were similar to those in control cells without exogenous PIP2.
Next, we tested for the effects of promoting breakdown of PIP2 by stimulating phospholipase C (PLC)-coupled 
-adrenergic and muscarinic receptors (3) with phenylephrine (40 µM) and carbachol (100 µM). Under these conditions there was no significant change of IMIC compared with predrug levels (phenylephrine: relative IMIC = 96 ± 4%, P = 0.35, n = 4; carbachol: relative IMIC = 103 ± 4%, P = 0.21, n = 5), suggesting that receptor stimulation in the presence of PIP2 resynthesis is ineffective in inducing rundown. We also wanted to test for the involvement of PLC by inhibiting the enzyme with U-73122 (10 µM) applied internally in ATP-loaded cells. We noted that U-73122 completely inhibited IMIC (n = 5; not illustrated), limiting its usefulness as a tool in assessing the role of PLC. Direct inhibition of TRPM7 channels by U-73122 has also been described (38, 44).
Finally, because the activity of the inward rectifier K+ current (IK1 or IKir2.1) is known to be modulated by PIP2 (17), we monitored the time course of both IMIC and IK1 in three cells dialyzed with ATP-free solution. IK1 was measured at patch rupture and at several points during the time course of IMIC. To measure IK1, IMIC was first blocked by introduction of extracellular divalent cations, and then cells were transiently exposed to the standard K+-containing external Tyrode solution. IK1, measured as inward current at –120 mV, decreased concurrently with the rundown of IMIC (not shown). At times near complete IMIC rundown, the currents had decreased to 5 ± 2% (IK1) and 6 ± 4% (IMIC) of their peak values. A similar parallel rundown of IKir2.1 and IMIC has also been observed in rat basophilic leukemia cells (21). The prevention of rundown by exogenous PIP2 and the parallel rundown of IMIC and IK1 support the view that PIP2 is necessary for MIC channel activity.
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The known affinity of ATP for Mg2+ raises the question of whether the observed rundown is not due to a simple change of intracellular Mg2+ on loading or depleting ATP in the cell. The rundown of IMIC in ATP-depleted cells could eventually be attributed to an inhibition by free Mg2+ previously bound to ATP. However, this is unlikely because the nonhydrolyzable ATP analog AMP-PNP, which binds Mg2+ like ATP (2), failed to prevent rundown. In addition, such a release of free Mg2+ would be rapidly diluted (35) by the bulk of low-Mg2+ or Mg2+-free pipette solutions. Thus our data are consistent with a direct role of ATP, independent of its ability to chelate Mg2+.
There has been some uncertainty about the role of intracellular ATP in regulating TRPM7 and related channels. Mg2+-nucleotides such as MgATP and MgGTP inhibit TRPM7 channels (32), but this inhibition has also been attributed entirely to free internal Mg2+ or other divalent cations (20). Consistent with a direct inhibition by divalent cations, our previous results (10, 48) showed that intracellular Mg2+ alone is effective in inhibiting MIC channels. Recent results indicate that binding of Mg2+-nucleotides on the kinase domain of TRPM7 modulates the inhibition induced by free Mg2+ (6). On the other hand, ATP enhances the TRPM7 current during channel activation (39). Our present data demonstrate that ATP hydrolysis is required for channel activity and therefore point to a role of kinases in regulating MIC channel activity.
TRPM6 and TRPM7 channels are known to possess kinase function, hence their designation as "chanzymes" (30), but the exact role of this function is uncertain. The TRPM7 kinase is modulated by Mg2+, and the channel protein can undergo autophosphorylation (40). Although the kinase function influences the sensitivity of the channel to Mg2+ or MgATP (42), neither the kinase nor the channel autophosphorylation is needed for channel function (27, 42). Nevertheless, in HEK-293 cells there is a cAMP-mediated modulation of the TRPM7 channel activity that requires the channel kinase (44). Our results show that stimulation of adenylate cyclase by forskolin failed to modulate cardiac MIC channels, despite being able to augment ICaL under similar conditions. Different auxiliary channel subunit composition or possible channel heteromultimerization may account for the differences in cAMP sensitivity among cell types.
TRPM7 interacts with isoforms of PLC (38), one of which (PLC) is regulated by tyrosine kinase. However, the functional implication of the interaction remains undetermined. Our results suggest that, among the classic protein kinases, only protein tyrosine kinase modulates cardiac MIC channel activity. Effects similar to those in cardiac myocytes have been obtained with tyrosine kinase modulation of the TRPM7-like current in brain microglia, where the regulation is proposed to occur via activation of PLC (18). In the present study, we did not investigate the signaling cascade involved in the modulation by tyrosine kinase, but the involvement of mechanisms similar to those proposed for microglia cannot be ruled out. Despite the modulation of MIC channels by tyrosine kinase, inhibition of protein tyrosine phosphatase could not prevent ATP depletion-related rundown. Therefore, the phosphorylation mediated by tyrosine kinase does not appear to be the main process underlying the activation of MIC channels, especially in preventing rundown.
The prevention of rundown in ATP-depleted cells by lipid phosphatase inhibition and the induction of rundown in ATP-loaded cells following the addition of drugs that deplete endogenous membrane phosphoinositides suggest the requirement of ATP in lipid metabolism modulating MIC channels. We speculated that cell membrane phospholipid depletion (especially PIP2), probably due to ongoing endogenous hydrolysis, could be the mechanism underlying IMIC rundown. The prevention of rundown in ATP-depleted cells in which exogenous PIP2 was added to the internal solution supports this hypothesis. In addition, the rundown of IMIC occurred in parallel with that of IK1, which is known to depend on PIP2 and lipid kinases (17). Although the effects of PIP2 antibody treatment were marginal (attributable to slow diffusion of the antibody as discussed above), altogether the results are consistent with the regulation of MIC channels by PIP2 and lipid kinases.
PIP2 regulates various ion transporters and channels (16). In cardiac cells, besides IK1, other inward rectifier K+ channels such as the muscarinic receptor-activated K+ channels are known to require PIP2 (17). Similarly, the activity of TRP channels such as TRPM7 (38) and TRPM8 (24) depend on PIP2. In contrast, PIP2 downregulates other members of TRP subfamilies, e.g., TRPV1 (4) and Drosophila TRP and TRPL (13). Thus MIC channels in cardiac myocytes belong to the group of channels modulated by PIP2. Local PIP2 is proposed to regulate ion channels by electrostatic modulation, either directly by binding to intracellular components of the channel or indirectly by altering interactions between channel and other regulatory units (7, 16). More work is still required to understand the mechanisms by which PIP2 modulates cardiac MIC channels.
We previously showed that MIC channels are pH sensitive and, based on their permeability, speculated that the channels may form a significant pathway for influx of divalent cations such as Ca2+ and Mg2+ (10). Acidosis and low intracellular ATP, and the subsequent loss of PIP2, associated with conditions such as ischemia would favor inhibition of MIC channels, resulting in reduced influx of Ca2+, Mg2+, and other cations. We therefore propose that channel sensitivity to ATP and PIP2 may contribute to limiting disturbances of ion homeostasis during metabolic stress conditions such as ischemia.
In conclusion, our results suggest that PIP2, likely generated by ATP-utilizing lipid kinases, is necessary for maintaining the activity of cardiac MIC channels. Although the exact role of cardiac MIC channels still remains to be established, our data are consistent with a role in linking ion homeostasis with cell metabolism. Additional evidence in support of a role for PIP2 regulation of TRPM7 (22) and of TRPV5 (23) has appeared recently.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Bagshaw C. ATP analogues at a glance. J Cell Sci 114: 459–460, 2001.
3. Brown JH, Buxton IL, and Brunton LL. Alpha 1-adrenergic and muscarinic cholinergic stimulation of phosphoinositide hydrolysis in adult rat cardiomyocytes. Circ Res 57: 532–537, 1985.
4. Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, and Julius D. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 411: 957–962, 2001.[CrossRef][Medline]
5. Chubanov V, Waldegger S, Mederos y Schnitzler M, Vitzthum H, Sassen MC, Seyberth HW, Konrad M, and Gudermann T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. Proc Natl Acad Sci USA 101: 2894–2899, 2004.
6. Demeuse P, Penner R, and Fleig A. TRPM7 channel is regulated by magnesium nucleotides via its kinase domain. J Gen Physiol 127: 421–434, 2006.
7. Dubyak GR. Ion homeostasis, channels, and transporters: an update on cellular mechanisms. Adv Physiol Educ 28: 143–154, 2004.
8. Fleig A and Penner R. The TRPM ion channel subfamily: molecular, biophysical and functional features. Trends Pharmacol Sci 25: 633–639, 2004.[CrossRef][Medline]
9. Gwanyanya A, Amuzescu B, Vereecke J, and Mubagwa K. Mg2+-inhibited cation channel in cardiac myocytes: dependence of activation on intracellular ATP and modulation by guanine nucleotide analogues (Abstract). Biophys J 88: 117A–118A, 2005.
10. Gwanyanya A, Amuzescu B, Zakharov SI, Macianskiene R, Sipido KR, Bolotina VM, Vereecke J, and Mubagwa K. Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation. J Physiol 559: 761–776, 2004.
11. Hanano T, Hara Y, Shi J, Morita H, Umebayashi C, Mori E, Sumimoto H, Ito Y, Mori Y, and Inoue R. Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. J Pharmacol Sci 95: 403–419, 2004.[CrossRef][ISI][Medline]
12. Hardie DG. Roles of protein kinases and phosphatases in signal transduction. Symp Soc Exp Biol 44: 241–255, 1990.[Medline]
13. Hardie RC. Regulation of TRP channels via lipid second messengers. Annu Rev Physiol 65: 735–759, 2003.[CrossRef][ISI][Medline]
14. He Y, Yao G, Savoia C, and Touyz RM. Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II. Circ Res 96: 207–215, 2005.
15. Hilgemann DW. Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers. Annu Rev Physiol 59: 193–220, 1997.[CrossRef][ISI][Medline]
16. Hilgemann DW, Feng S, and Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE 2001: RE19, 2001.[Medline]
17. Huang CL, Feng S, and Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G. Nature 391: 803–806, 1998.[CrossRef][Medline]
18. Jiang X, Newell EW, and Schlichter LC. Regulation of a TRPM7-like current in rat brain microglia. J Biol Chem 278: 42867–42876, 2003.
19. Kim BJ, Lim HH, Yang DK, Jun JY, Chang IY, Park CS, So I, Stanfield PR, and Kim KW. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity. Gastroenterology 129: 1504–1517, 2005.[CrossRef][ISI][Medline]
20. Kozak JA and Cahalan MD. MIC channels are inhibited by internal divalent cations but not ATP. Biophys J 84: 922–927, 2003.
21. Kozak JA, Kerschbaum HH, and Cahalan MD. Distinct properties of CRAC and MIC channels in RBL cells. J Gen Physiol 120: 221–235, 2002.
22. Kozak JA, Matsushita M, Nairn AC, and Cahalan MD. Charge screening by internal pH and polyvalent cations as a mechanism for activation, inhibition, and rundown of TRPM7/MIC channels. J Gen Physiol 126: 499–514, 2005.
23. Lee J, Cha SK, Sun TJ, and Huang CL. PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2+. J Gen Physiol 126: 439–451, 2005.
24. Liu B and Qin F. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate. J Neurosci 25: 1674–1681, 2005.
25. Macianskiene R, Matejovic P, Sipido K, Flameng W, and Mubagwa K. Modulation of the extracellular divalent cation-inhibited non-selective conductance in cardiac cells by metabolic inhibition and by oxidants. J Mol Cell Cardiol 33: 1371–1385, 2001.[CrossRef][ISI][Medline]
26. Macianskiene R, Moccia F, Sipido KR, Flameng W, and Mubagwa K. Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells. Am J Physiol Heart Circ Physiol 282: H1879–H1888, 2002.
27. Matsushita M, Kozak JA, Shimizu Y, McLachlin DT, Yamaguchi H, Wei FY, Tomizawa K, Matsui H, Chait BT, Cahalan MD, and Nairn AC. Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1. J Biol Chem 280: 20793–20803, 2005.
28. Meyers R and Cantley LC. Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase. J Biol Chem 272: 4384–4390, 1997.
29. Monteilh-Zoller MK, Hermosura MC, Nadler MJ, Scharenberg AM, Penner R, and Fleig A. TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. J Gen Physiol 121: 49–60, 2003.[CrossRef][ISI][Medline]
30. Montell C. Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes. Curr Biol 13: R799–R801, 2003.[CrossRef][ISI][Medline]
31. Mubagwa K, Stengl M, and Flameng W. Extracellular divalent cations block a cation non-selective conductance unrelated to calcium channels in rat cardiac muscle. J Physiol 502: 235–247, 1997.
32. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, and Fleig A. LTRPC7 is a Mg·ATP-regulated divalent cation channel required for cell viability. Nature 411: 590–595, 2001.[CrossRef][Medline]
33. Nakanishi S, Catt KJ, and Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci USA 92: 5317–5321, 1995.
34. Perraud AL, Knowles HM, and Schmitz C. Novel aspects of signaling and ion-homeostasis regulation in immunocytes. The TRPM ion channels and their potential role in modulating the immune response. Mol Immunol 41: 657–673, 2004.[CrossRef][ISI][Medline]
35. Pusch M and Neher E. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch 411: 204–211, 1988.[CrossRef][ISI][Medline]
36. Ramsey IS, Delling M, and Clapham DE. An introduction to TRP channels. Annu Rev Physiol 68: 619–647, 2006.[CrossRef][ISI][Medline]
37. Ruhlmann A and Nordheim A. Effects of the immunosuppressive drugs CsA and FK506 on intracellular signalling and gene regulation. Immunobiology 198: 192–206, 1997.[ISI][Medline]
38. Runnels LW, Yue L, and Clapham DE. The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat Cell Biol 4: 329–336, 2002.[ISI][Medline]
39. Runnels LW, Yue L, and Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 291: 1043–1047, 2001.
40. Ryazanova LV, Dorovkov MV, Ansari A, and Ryazanov AG. Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel. J Biol Chem 279: 3708–3716, 2004.
41. Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, and Konrad M. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 31: 166–170, 2002.[CrossRef][ISI][Medline]
42. Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, and Scharenberg AM. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 114: 191–200, 2003.[CrossRef][ISI][Medline]
43. Suh BC and Hille B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15: 370–378, 2005.[CrossRef][ISI][Medline]
44. Takezawa R, Schmitz C, Demeuse P, Scharenberg AM, Penner R, and Fleig A. Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain. Proc Natl Acad Sci USA 101: 6009–6014, 2004.
45. Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, and Hoenderop JG. TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem 279: 19–25, 2004.
46. Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, and Sheffield VC. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet 31: 171–174, 2002.[CrossRef][ISI][Medline]
47. Wiedemann C, Schafer T, and Burger MM. Chromaffin granule-associated phosphatidylinositol 4-kinase activity is required for stimulated secretion. EMBO J 15: 2094–2101, 1996.[ISI][Medline]
48. Zakharov SI, Smani T, Leno E, Macianskiene R, Mubagwa K, and Bolotina VM. Monovalent cation (MC) current in cardiac and smooth muscle cells: regulation by intracellular Mg2+ and inhibition by polycations. Br J Pharmacol 138: 234–244, 2003.[CrossRef][ISI][Medline]
This article has been cited by other articles:
|
|
 |
|
  R. M. Touyz Transient receptor potential melastatin 6 and 7 channels, magnesium transport, and vascular biology: implications in hypertension Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1103 - H1118. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
, 1.8 mM Ca2+, 0.9 mM Mg2+). Other traces were taken at maximum values (
) and at 50 min after patch rupture (
, dashed line) in the presence of extracellular divalent cations (A and C) or in their absence (0 mM Ca2+, 0 mM Mg2+; B and D). Note the decreased IMIC in ATP-depleted cells after 50-min cell dialysis. ATPi, internal ATP concentration (in mM).
