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Cardiovascular Research 2007 73(3):531-538; doi:10.1016/j.cardiores.2006.11.023
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Copyright © 2006, European Society of Cardiology

TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells

Marie Demiona, Patrick Boisa, Pierre Launayb and Romain Guinamarda,*

aInstitut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Université de Poitiers, F-86022 Poitiers Cedex, France
bInserm, U699 Equipe Avenir, Paris, F-75018, France

* Corresponding author. CNRS UMR 6187, Université de Poitiers, 40 av. du recteur Pineau, 86022 POITIERS Cedex, France. Tel.: +33 5 49 45 37 47; fax: +33 5 49 45 40 14. Email address: romain.guinamard{at}univ-poitiers.fr

Received 18 September 2006; revised 30 October 2006; accepted 17 November 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: A calcium-activated nonselective cation channel (NSCCa) has been recently described in several cardiac preparations. This channel is over-expressed in models of ventricular hypertrophy showing electrophysiological perturbations of heart activity, including occurrence of spontaneous activity. While these perturbations are currently attributed to a modification of the pacemaker If current activity, arguments are also in favor of participation of an NSCCa. Similarly, the NSCCa may be expressed in specialized pacemaker cells, i.e. sino-atrial node (SAN) cells. The aim of the present study was to detect such current in mouse pacemaker cells.

Methods: The inside-out configuration of the patch-clamp technique was used in freshly isolated SAN cells from adult mice. Also, RT-PCR and Western-blotting studies were used to probe for TRPM4 mRNA and protein expression.

Results: In these cells, an NSCCa activity was detected. The channel is voltage dependant with a conductance of 20.9±0.5 pS (n=11). It is equally permeable for Na+ and K+ but does not conduct Ca2+. It is activated by rise in intracellular calcium concentrations and blocked by intracellular ATP (0.5 mmol/L). Also, as a new property in cardiac cells, the channel is activated by internal application of phosphatidylinositol 4,5-bisphosphate (10 µM). It is reversibly inhibited by flufenamic acid and glibenclamide. This channel shows the hallmarks of the TRPM4 molecule, a member of the TRP melastatin subfamily. We confirm the expression of this TRP channel on SAN cells by Western blotting and RT-PCR and validate that TRPM4 is glibenclamide sensitive.

Conclusion: TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.

KEYWORDS Arrhythmias; Ion channels; Single channel; Sinus node


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In several cardiac preparations, we recently described a calcium-activated nonselective cation channel (NSCCa) (see [1] for review). This NSCCa current is characterized by voltage dependence, a single channel conductance close to 20 pS, an absence of discrimination between monovalent cations and no permeability to Ca2+. It is activated by intracellular calcium ions and the Protein Kinase C (PKC) pathway but inhibited by cytosolic ATP and flufenamic acid. In human atrial cardiomyocytes, Guinamard et al. [2] described the presence of such current. It is suspected to play a role in the macroscopic calcium-activated nonselective cationic current implicated in genesis of the transient inward current (Iti) responsible for Delayed After Depolarizations (DADs). At the ventricular level, while only slightly expressed on normotensive rat ventricular cells, its functional expression increases in a model of dedifferentiating ventricular cells in culture [3]. Also, its detection was frequent in freshly isolated ventricular myocytes from spontaneously hypertensive rats (SHR) that develop cardiac hypertrophy [4]. These are two models of cardiac remodeling associated with occurrence of arrhythmias [5]. In these models, modifications of the electrical activity are actually explained by an increase in pacemaker current (If) and transient calcium current (ICaT) density associated with a decrease in the transient outward potassium current (Ito) [6,7]. Thus, these cells acquire in part a pattern of channel expression closer to that of normal pacemaker cells, which express both channels If and ICaT. As the NSCCa is also over-expressed during ventricular remodeling, one can postulates that it participates to the triggering and/or dysfunction of spontaneous activity in addition to If and ICaT. By analogy, the NSCCa may be expressed in specialized pacemaker cells i.e. sino-atrial node (SAN) cells.

The aim of the present study was to investigate the presence of an NSCCa on SAN cells. Using the inside-out configuration of the patch-clamp technique, we analyzed the electrophysiological and regulatory properties of an NSCCa on mouse SAN cells. As the recently cloned TRPM4 [8] protein is the expected molecular correlate, we compared its electrophysiological properties to those of the SAN channel and looked for messenger RNA and immunodetection of TRPM4 protein on those cells.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Mouse sino-atrial cells isolation
Adult female Swiss mice (Mus musculus; 4–8 weeks) were treated and sacrificed in agreement with the guide for the care and use of laboratory animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). SAN cells were isolated using methods adapted from Difrancesco et al. [9] and Mangoni and Nargeot [10]. Beating heart was quickly removed via thoracotomy and transferred to a Tyrode solution containing 2.5 I.U./mL Heparin. The SAN region was excised and finely minced. Tissue strips were transferred into a "Tyrode low Ca2+/EGTA" solution with 0.5% bovine serum albumin (BSA, Sigma Aldrich) during 4 min, then into the same solution without EGTA and containing 52 I.U./mL collagenase (type IA, Sigma Aldrich), 0.33 I.U./mL protease (type XXIV, Sigma Aldrich), 0.2 I.U./mL elastase (type IV, Sigma Aldrich) and 0.5% BSA. Digestion was carried out for 8–12 min (30 °C). Samples were transferred into a "Kaftbrühe" (KB) medium [11] with 0.3% BSA and gently triturated using a fire-polished Pasteur pipette. Cells were seeded in 35 mm Petri dishes and superfused in a bath solution used for electrophysiological experiments. For electrophysiology, cells were used within 6 h after isolation.

2.2. RNA isolation
Isolated SAN tissues from ten mice were pooled. RNA was isolated using trizol reagent (Eurobio) followed chloroform extraction and isopropanol precipitation. RNA integrity was evaluated by ethydium bromide staining on a 1% agarose gel. RNA samples were stored at –80 °C.

2.3. RT-PCR
50 µL of total RNA was added to 60 µL of reaction mixture (100 mmol/L Tris–HCl pH 8.3, 150 mmol/L KCl, 6.25 mmol/L MgCl2, 20 mmol/L DTT, 2 mmol/L dNTPs and 13 µg Random Primer p(dN)6; Invitrogen). RNA were denatured at 65 °C during 2 min and then added to 8 U RNAse inhibitor and 160 U M-MLV reverse transcriptase (Invitrogen) to 125 µL final volume. cDNA was synthesized at 37 °C for 1 h and remaining enzymes were heat deactivated (100 °C, 2 min) and then added with 125 µL sterile water. 2.5 µL of cDNA was added to 22.5 µL of reaction mixture (50 mmol/L KCl, 10 mmol/L Tris–HCl pH 9, 3.5 mmol/L MgCl2, 0.25 dNTPs and 0.4 µM forward and reverse primers) with 1.25 U of Taq Polymerase (Invitrogen) and were amplified for 35 cycles. The amplification profile involved denaturation at 94 °C for 30 s, primers annealing for 30 s and primers extension at 72 °C for 30 s. After the last cycle, samples were incubated at 72 °C for 10 min to extend incomplete products. The PCR products were analyzed on 1% agarose gel. Negative controls were performed without RNA.

2.4. Western blotting
Total proteins were isolated from SAN tissues from 10 mice and protein concentration determined by Bio-Rad DC protein assay methods (Bio-Rad). 20 µg of protein was separated on 8% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with TBS (50 mmol/L Tris–HCl, 500 mmol/L NaCl, pH 7.5) containing 0.1% Tween and 3% BSA. The membrane was incubated with primary antibody overnight at 4 °C, washed and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. A goat anti rabbit secondary antibody conjugated with HRP was used for revelation.

2.5. Cell culture
Tetracycline-inducible HEK-293 Flag-TRPM4-expressing cells (from Launay et al. [8]) were cultured at 37 °C/5% CO2 in DMEM (Dulbecco's Modified Eagle Medium, Cambrex) supplemented with 10% Fetal Bovine Serum and 2 mmol/L glutamine. The medium was supplemented with S-Blasticidin (5 µg/mL; Invitrogen) and Zeocin (0.4 mg/mL; Invitrogen). For all experiments, cells were resuspended in media containing 1 µg/mL tetracycline (Sigma) 18–22 h before experiments.

2.6. Solutions and chemicals
For cell dissociation, the Tyrode solution contained in mmol/L: 140 NaCl, 5.4 KCl, 1.8 MgCl2, 1.8 CaCl2, 10 glucose and 10 HEPES, pH was adjusted to 7.4 with NaOH. The "Tyrode low Ca2+/EGTA" solution contained in mmol/L: 140 NaCl, 5.4 KCl, 1.8 MgCl2, 10 glucose, 20 Taurine, 5 HEPES and 0.1 EGTA, pH was adjusted to 7.2 with NaOH.

For electrophysiological recordings, the standard bath solution contained in mmol/L: 140 NaCl, 4.8 KCl, 1.2 MgCl2, 10 glucose, 10 HEPES and 0.1 µM CaCl2. Pipette solution contained in mmol/L: 145 NaCl, 1.2 MgCl2, 10 glucose, 10 HEPES and 1 CaCl2. Perfusion solution usually contained in mmol/L: 145 NaCl, 1.2 MgCl2, 10 glucose, 10 HEPES and 1 CaCl2. Internal Ca2+ concentrations below 10 µM were determined with a combination of CaCl2 and Ca-EGTA buffers or addition of EGTA [2]. Low NaCl solutions contained 42 or 14 mmol/L NaCl (no KCl included) and were supplemented with sucrose to maintain osmolarity. When specified, 145 mmol/L NaCl was replaced by 145 mmol/L KCl. Ca2+ selectivity was determined using a solution containing in mmol/L: 100 CaCl2, 10 glucose and 10 HEPES. External solutions (bath and pipette) were adjusted to pH 7.4. The pH of perfused solutions (inside of the membrane) was adjusted to 7.2.

Chemical products were from Sigma Aldrich. DiC8-PIP2 was purchased from Echelon Biosciences. DiC8-PIP2 and glibenclamide were dissolved in DMSO to a final DMSO ratio of 0.1% that has no effect on channel activity at this concentration.

2.7. Measurements
Single-channel currents from patches of isolated SAN cells were recorded under voltage clamp with an RK 400 (Biologic, Claix, France) patch-clamp amplifier using the inside-out configuration of the patch-clamp technique [12]. Liquid junction potentials were determined using a pipette containing 2.7 M KCl, which allowed the zero-current voltage deflection to be monitored and confirmed by the JPCalc program [13]. Applied potentials (Vm=Vbath–Vpipette) were corrected accordingly. Currents due to the migration of cations from the inner to the outer surface of the membrane were positive and were registered as upward deflections in single-channel current tracing. Experiments were conducted at room temperature (20–25 °C).

2.8. Data analysis
Signals for analysis were stored on digital audio tapes, played back through a filter (Bessel model 902LPF, Frequency Devices, Inc., Haverhill MA, USA) and digitized at 1 KHz using digidata 1200A analog-digital interface and Fetchex software (version 6.02; Axon Instruments). Currents were analyzed with Bio-patch software (version 3.30; Biologic), generating amplitude histograms for the construction of I–V curves and the estimation of open probability (Po). Relative permeabilities and associated reversal potentials for current flow (Vrev) were estimated by fitting experimental measurements to the Goldmann–Hodgkin–Katz equation [2]. Analysis was performed using the Origin Software (version 5.0, Microcal, Northampton MA, USA).

Experimental results are reported as mean±S.E.M. A paired Student's test was applied when Po was determined under different conditions on the same cell. An analysis of variance (ANOVA) was used to compare conductances and reversal potentials under different conditions. Statistical significance was accepted for values of P<0.05.

In the figures, statistical significances were indicated as below:

ns: nonsignificant

*: P<0.05

**: P<0.01

***: P<0.001.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
We recently reported the presence of a calcium-activated nonselective cation channel in excised inside-out patches from adult rat cardiomyocytes [3] and freshly isolated human atrial cardiomyocytes [2].

In order to investigate the presence of this channel in mouse SAN cells, single channel currents were recorded from freshly isolated cells. Pacemaker cells were selected for their typical elongated shape described by Mangoni and Nargeot [10]. A typical cell is shown in Fig. 1. Channel activity was recorded using the inside-out configuration of the patch-clamp technique with 145 mmol/L NaCl standard solution on both sides of the membrane. Fig. 2A illustrates channel activity under these ionic conditions as a function of membrane potential. The corresponding current–voltage relationship (Fig. 2B) was linear with a slope conductance estimated at 20.9±0.5 pS (n=11) and a reversal potential at –0.4±1.1 mV. The open probability values determined at various membrane potentials indicates that channel activity significantly increases with depolarization (Fig. 2C, n=4). This current was observed in 24.3% of patches with a mean number a channel/patch of 0.28±0.05 pS (n=94).


Figure 1
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  		Fig. 1 Typical pacemaker cell. Phase-contrast micrograph of freshly isolated pacemaker mouse sinus node cell. Note the characteristic shape of the cell.

 

Figure 2
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  		Fig. 2 Conductive properties of the non-selective cation channel. A. Single-channel tracings recorded at various voltages from an inside-out patch. Pipette contained 145 mmol/L NaCl standard solution with CaCl2 1 mmol/L and perfusion 145 mmol/L NaCl standard solution with CaCl2 1 mmol/L. Vm corresponds to the membrane potential. Dotted lines indicate the current level of closed channels. B. Current–voltage relationship under the same ionic conditions as in A. Data points (mean from 11 different patches for each point) were fitted by linear regression, yielding the following values: slope conductance (g), 20.9±0.5 pS; reversal potential (Vrev), –0.4±1.1 mV (n=11). C. Voltage dependence of open probability (Po). In 4 experiments similar to that shown in A (145 mmol/L NaCl standard solution with CaCl2 1 mmol/L in both side of the membrane), Po was determined at various voltages using amplitude histograms. D. Single-channel current tracings recorded at various voltages from an inside-out patch. Pipette 145 mmol/L NaCl, perfusion 42 mmol/L NaCl. E. Current–voltage relationship under the same conditions as in D averaged from 4 patches. Data were fitted by the GHK equation, giving values of Vrev=+25.5±4 mV and PNa/PCl=12.9. F. Current–voltage relationships in the presence of 145 mmol/L KCl (open circles, g=25.3±0.3 pS, Vrev=–0.7±2.3 mV, PNa/PK=0.97, n=4) or in the presence of 100 mmol/L CaCl2 (black squares), data points were fitted by the GHK equation (Vrev=+60±3.3 mV, PNa/PCa=11, n=4).

 
3.1. Selectivity
The ionic selectivity was investigated in inside-out patches by changing the ionic composition at the cytoplasmic side. Reducing the NaCl concentration from 145 mmol/L to 42 mmol/L or 14 mmol/L on the inner side of the membrane shifted the reversal potential to more positive voltages (Fig. 2D, E). Fitting of the Goldmann–Hodgkin–Katz (GHK) equation to the data yielded mean conductance values of 24.1±2.4 pS (n=4) and 21.8±1 pS (n=5) (not significantly different from symmetrical conditions) and reversal potential values of +25.5±4 mV (n=4) and +40.8±4.3 mV (n=5), for 42 mmol/L NaCl and 14 mmol/L NaCl solutions, respectively. The corresponding permeability ratios PNa/PCl are 12.9 and 12, respectively.

Monovalent cations selectivity was assessed by replacing 145 mmol/L NaCl in the perfusing solution by 145 mmol/L KCl. Channel conductance (25.3±0.3 pS) and Vrev (–0.7±2.3 mV, n=4) were not significantly different from that of control (145 mmol/L NaCl) (Fig. 2F). The corresponding permeability ratio (PNa/PK) was 0.97. To test the calcium permeability of the channel, the cytoplasmic side was bathed in a solution containing 100 mmol/L CaCl2. Under these conditions, we detected inward currents but we failed to detect outward currents. The channel displayed a conductance of 24.6±0.5 pS. The reversal potential was estimated at +60±3.3 mV (n=4), corresponding to a PNa/PCa=11 (Fig. 2F). The results indicate that the channel primarily conducts monovalent Na+ as well as K+ ions, but not calcium.

3.2. Channel regulation
As reported on several tissues (see [14] for review), the nonselective cation channel is dependent upon the cytosolic calcium concentration. Fig. 3A clearly shows that reducing the calcium concentration from 1 mmol/L to 10 µmol/L on the cytoplasmic side of an excised inside-out patch decreased channel activity. The open probability of active channels determined at +40 mV is plotted in Fig. 3B as a function of internal Ca2+ concentration, rising from 1 nmol/L to 1 mmol/L (n=2–7). Channel openings were seen at [Ca2+]i≥1 µmol/L. Fitting of the Hill equation to the experimental data points yielded an apparent dissociation constant (Kd) of 154 µmol/L and a Hill coefficient of 0.80.


Figure 3
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  		Fig. 3 Channel regulation. A. Single channel currents recorded from an inside-out patch showing the increase of the channel activity with rising [Ca2+]i (Vm=+40 mV, pipette and perfusion contained 145 mmol/L NaCl). ‘c’ and ‘o’ indicate the current levels for the close and the open states of the channel respectively. B. Values of Po (mean±SEM) at various [Ca2+]i (Vm=+40 mV, n=2–7). C. Single channel currents recorded from an inside-out patch showing the reversible inhibition of channel activity by 0.5 mmol/L ATP (Vm=+40 mV). D. Corresponding amplitude histogram showing the reversible effect of ATP on channel open probability (n=6). E. Single channel tracing recorded from an inside-out patch showing the increase of channel activity when PIP2 10 µmol/L was perfused at the inner side of the membrane (10–6 M [Ca2+]i, Vm=+40 mV; pipette and perfusion contained 145 mmol/L NaCl). F. Corresponding amplitude histogram showing the increase of open probability with PIP2 (n=5), (Vm=+40 mV).

 
The cardiac nonselective cation channels have also been shown to be sensitive to cytosolic adenine nucleotides [2,3]. This effect of internal ATP was assessed using the inside-out configuration of the patch-clamp technique. Fig. 3C and D show that addition of 0.5 mmol/L ATP-Na2 to the cytoplasmic side of an inside-out patch reversibly suppresses channel activity (Po=0.01±0.01 compared to 0.58±0.17 in control; n=6; Vm=+40 mV).

Recently, it was demonstrated that PIP2 is a strong positive regulator of a calcium-activated nonselective cationic channel, the TRPM4 protein [15,16]. We observed the same properties on the SAN NSCCa channel. The perfusion of 10 µmol/L of PIP2 significantly increased the Po of NSCCa even in 10–6 mol/L [Ca2+]i (Fig. 3E, F). Po rose from 0.17±0.04 in control to 0.62±0.12 with PIP2 (n=5; P<0.01; Vm=+40 mV; [Ca2+]i 10–6 mol/L).

3.3. Pharmacology
Flufenamic acid, a non-steroidal anti-inflammatory drug has been reported to block NSC channels in several preparations including cardiac cells [4,17]. Fig. 4 illustrates the reversible action of this compound on SAN NSCCa channel. The perfusion of 0.1 mmol/L flufenamic acid in the bath clearly reduced channel activity to 31.7±15.2% that of control (Vm=+40 mV, n=4).


Figure 4
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  		Fig. 4 Channel inhibition by flufenamic acid and glibenclamide. A. and B. Single channel currents recorded from an inside-out patch illustrating the reversible blocking effect of 0.1 mmol/L flufenamic acid (FA) or 0.1 mmol/L glibenclamide (Glib.) (Vm=+40 mV). C. Po in the absence (control) or in the presence of 0.1 mmol/L flufenamic acid or 0.1 mmol/L glibenclamide (Vm=+40 mV, n=4 and 3, respectively).

 
The ATP effect previously described could suggest a sulphonylurea sensitivity of the channel. In that sense, the inhibition of NSCCa channel activity by glibenclamide, a hypoglycemic agent, has been reported in human atrial cardiomyocytes [2].Fig. 4B and C show the reversible action of 100 µmol/L of this compound on channel activity that fall to 40±16.7% that of control in presence of glibenclamide (n=3).

3.4. Glibenclamide sensitivity of TRPM4 in HEK 293 cells
The electrophysiological and regulatory properties of the SAN NSCCa were also described for the TRPM4 protein [8,18] except for the glibenclamide sensitivity that was never reported for TRPM4 expressed in cell lines. To investigate if this property is specific for cardiac preparations or can be enlarge to the TRPM4 protein, we tested the efficiency of this compound on heterologously expressed TRPM4 using the inside-out configuration of the patch-clamp technique.

As illustrated in Fig. 5, when expressed in HEK 293 cells, TRPM4 showed a linear current–voltage relationship with a slope conductance of 23.5±1 pS (n=5, not significantly different from the NSCCa recorded in mouse SAN cells, P =0.54) and a reversal potential at 0.91±1.9 mV (P=0.53). Application of glibenclamide at 100 µmol/L at the inner face of the membrane (Vm=–40 mV) significantly decreased TRPM4 activity to 22% that of control (n=5, P<0.001). This inhibition is similar to that of SAN NSCCa. It is the first demonstration that TRPM4 is glibenclamide sensitive.


Figure 5
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  		Fig. 5 Pharmacological analysis of TRPM4 bearing HEK-293 cells A. Single-channel tracings recorded at various voltages from an inside-out patch from TRPM4 transfected HEK 293 cells. Pipette and bath contained 145 mmol/L NaCl standard solution (CaCl2: bath 10–4 mol/L, pipette 1 mmol/L). Current–voltage relationship under the same ionic conditions (g=23.5±15 pS, Vrev=0.9±1.9 mV, n=5). B. Single channel currents recorded from an inside-out patch illustrated the reversible blocking effect of 0.1 mmol/L glibenclamide (Vm=–40 mV). As several identical channels are present in the patch membrane, labels ‘c’, ‘3’ and ‘6’ indicate the current levels when all channels are closed or when 3 or 6 are open. The amplitude histogram showed the Po in absence (control) or presence of 0.1 mmol/L glibenclamide (Vm=–40 mV, n=5).

 
3.5. TRPM4 expression in SAN tissue
As reported previously in human atrial cardiomyocytes [2], the SAN NSCCa showed the electrophysiological (conductance, selectivity and pharmacology) and regulatory (Ca2+, ATP) properties described for the human TRPM4 protein expressed in HEK 293 [8,18]. We thus performed experiments to probe for TRPM4 mRNA and protein expression from mouse SAN samples. The TRPM4 transcript was detected by RT-PCR. Fig. 6A shows the presence of the TRPM4 transcript using different pairs of primers. Also, the TRPM4 protein was detected using western blot technique in SAN tissue. Fig. 6B shows a band at the expected molecular weight.


Figure 6
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  		Fig. 6 TRPM4 mRNA and protein detection in mouse SAN tissue. A. Representative agarose gel from RT-PCR experiments. From left to the right: Lane 1 shows the marker of weight. Lanes 2 and 3 show the result of RT-PCR realized with SRP primers, lanes 4–5 and 6–7 show the result of RT-PCR realized with 2 TRPM4 primers pairs (forward 4+reverse 9 and forward 6+reverse 9, see Table 1 for sequences) (H20=negative control, SAN=RNA extraction of SAN from ten mice). B. Representative western blots obtained with anti-TRPM4 antibody. Sample of SAN tissue from ten mice was pooled for protein expression. From left to the right: Lane 1 shows the marker of weight. A band at the expected molecular mass of ~130 kDa is obtained in intestine (lane 2) with total protein to 175 µg and in SAN tissue (lane 3) with total protein to 205 µg.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. A calcium-activated nonselective cationic channel in mouse SAN cells corresponding to TRPM4
Using a cell-free configuration of the patch-clamp technique, the present study shows the existence of a Ca2+-activated nonselective cation channel in freshly isolated mouse sino-atrial node cells. Its properties are similar to that of Ca2+-activated NSC channels present in excitable and non-excitable tissues such as heart, epithelia, neurons, exocrine tissues and cells from sensory organs (see [1,14] for review). Their common characteristics are, voltage dependence, single channel conductance close to 20 pS, lack of discrimination between monovalent cations, low permeability to Ca2+ ions, sensitivity to intracellular calcium, inhibition by cytosolic ATP and blocking by flufenamic acid. Strong evidences support the idea that the TRPM4 protein is the molecular correlate of the current.

The transient receptor potential (TRP) channel family has been cloned in the last ten years. It is divided in three main subfamilies: TRPC for "canonical", TRPV for "vanilloid-receptor-like" and TRPM for "melastatin-like". While most of these channels are Ca2+ permeable, TRPM4 does not conduct Ca2+. Its selectivity sequence is Na+=K+>>Ca2+, Cl–. Moreover, TRPM4 has the biophysical properties of the NSCCa channel described here. TRPM4 is a calcium-activated nonselective cation channel with a unitary conductance close to 25 pS [8]. It is activated by internal calcium with a Kd of calcium activation close to 10 µmol/L [18]. It is voltage dependent [18,19] and blocked by internal ATP in the range of 10 µmol/L [20]. TRPM4 is also activated by intracellular PIP2 [15,16] and inhibited by flufenamic acid [21]. TRPM4 transcript is expressed in many tissues, including mouse and human hearts [2,18].

Moreover, we described here the blocking of the functional TRPM4 protein by the sulfonylurea inhibitor glibenclamide. This last point is of high interest. Indeed, glibenclamide is known to be effective on ATP binding cassette proteins (ABC proteins). These are, for example, the CFTR protein and sulfonylurea receptor [22,23]. Similarly to these proteins, TRPM4 holds two ABC transporters signature like motifs, consistent with the effect of glibenclamide [24]. Thus, this may be an interesting way to investigate in the design of molecular tools specific to TRPM4.

4.2. Channel regulation
Calcium sensitivity is probably the most relevant regulatory property of the TRPM4 channel. The Kd for channel activation by calcium was estimated in the range of 150 µmol/L in SAN. That is tenfold higher than the values of 10 µmol/L estimated in other cardiac preparations [2–4] but in the range of the value (370 µmol/L) reported in the inside-out configuration for TRPM4 expressed in HEK 293 cells [25]. Although that is a high level regarding the physiological Ca2+ concentration, it was shown in HEK 293 cells [25] that the Kd was close to 1 µmol/L in whole-cell recordings. This discrepancy with results obtained in the inside-out configuration would be explained by loss of intracellular regulators such as the calcium–calmodulin complex in this last configuration. Thus, Ca2+ sensitivity is probably underestimated in inside-out recordings. It was also reported by Zhang et al. [15] that TRPM4 activation by intracellular PIP2 was effective by shift of the calcium sensitivity in lower concentrations. Here, we showed that perfusion of 10 µmol/L of PIP2 at the inner face of the membrane significantly increase Po of the channel even in threshold calcium concentration for channel detection (10–6 mol/L). This finding suggests that the level of calcium required to activate TRPM4 may be sufficient in mouse SAN cells, at least when PIP2 is present.

4.3. Physiologic and pathophysiologic TRPM4 impact in SAN
The contribution of the NSCCa current to cardiac physiology is not yet understood, in particular because very few reports are available about macroscopic SAN NSCCa. Vinogradova et al. [26] showed in rabbit SAN cells that during the later part of the diastolic depolarization (DD), the local increase in submembrane [Ca2+] generates an Na+ inward current via Na+–Ca2+ exchange (NCX), enhancing the DD slope and modulating the occurrence of the next action potential. Although this current was attributed mainly to the NCX, it is conceivable that the NSCCa also contributes to this Ca2+-dependant inward Na+ current. Indeed, Sanders et al. [27] suggested the contribution of other depolarizing ionic components that were identified as being dependent on sarcoplasmic reticulum Ca2+ release in guinea pig SAN. However, it is not yet known if the calcium level reached at this moment is sufficiently high to produce the activation of the TRPM4 current.

According to its activation by intracellular Ca2+ and inhibition by intracellular ATP, it is plausible that the NSCCa current develops in the case of a pathophysiologic condition such as ischemia/reperfusion, when cell metabolism is beginning to be impaired and produces a deregulation of the calcium transient. Moreover, it could be involved in sinus tachyarrhythmias caused by cardiac steroids (digitalis) intoxication where the intracellular Ca2+ concentration in SAN increased via the NCX [28].

With the aim of evaluating whether the current could have an impact at the cellular level, it is possible to estimate the maximal whole-cell conductance of the TRPM4 current, taking in account that the mean number of channel/patch was 0.28, that the mean mouse SAN cell capacitance is 48 pF [29], and that the specific capacity of a biological membrane is 0.01 pF/µm2. By using these parameters, we estimated that around 420 channels are expressed in a single cell, providing a maximal conductance of 10 nS. This estimation supports the statement that TRPM4 is able to produce a significant current in the SAN cell. However, the physiological significance of this estimated value is difficult to evaluate and should be considered cautiously as channel activation is dependant on several parameters such as voltage, Ca2+, and ATP.

Unfortunately, in the absence of specific pharmacological tools, it is difficult to clearly identify the NSCCa at the whole-cell level and also evaluate its contribution to the action potential and heart beating. Nevertheless, our study identifies an additional channel that is expressed in mouse SAN cells. This protein could be regarded as a new therapeutic target in the control of heart rhythm.


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  		Table 1 Sequences for mRNA TRPM4 amplifications

 


   Acknowledgements
 
The authors thank Jean-François Faivre for technical help and helpful comments on the manuscript and Elise Mok for editing the manuscript. Marie Demion holds a PhD fellowship from the French Ministère de l'Enseignement et de la Recherche.


   Notes
 
Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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