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Cardiovascular Research 2003 58(1):76-88; doi:10.1016/S0008-6363(02)00858-1
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Copyright © 2003, European Society of Cardiology

A polycystin-2-like large conductance cation channel in rat left ventricular myocytes

Tilmann Volk*, Alexander Peter Schwoerer, Susanne Thiessen, Jobst-Hendrik Schultz and Heimo Ehmke*

Institut für Physiologie, Universitätskrankenhaus Hamburg-Eppendorf, Universität Hamburg, Martinistraße 52, 20246 Hamburg, Germany ehmke{at}uke.uni-hamburg.de

* Corresponding authors. Tel.: +49-40-42803-9615 (T. Volk); +49-40-42803-3183 (H. Ehmke); fax: +49-40-42803-9299. volk{at}uke.uni-hamburg.de

Received 2 July 2002; accepted 17 December 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objective: Several members of the PKD gene family (PKD2, PKDL and PKD2L2) are expressed in the heart. Polycystin-2 and its homologues, which are encoded by these genes, have recently been shown to form Ca2+-regulated nonselective cation channels in heterologous expression systems. Previously, large conductance nonselective cation channels (LCC) have been described in cardiomyocytes, however, their molecular identity remained obscure. We therefore examined whether LCCs may be formed by polycystins. Methods: Myocytes isolated from the rat left ventricle were investigated by the whole-cell patch-clamp technique and single-cell RT-PCR. Results: Application of 10 mM caffeine to the bath solution to increase the intracellular Ca2+ concentration led to activation of LCC in 56% of the myocytes investigated (total n = 651), in ~10%, more than three LCCs were detected. The single channel conductance was ~300 pS for monovalent cations and the channel was relatively nonselective for the monovalent cations Na+, K+, Li+, and Cs+ and also permeable for the divalent cations Ca2+ and Ba2+, but impermeable for NMDG+ and Cl–. Amiloride (IC50=131±1.1 µM) and millimolar concentrations of the trivalent cations Gd3+ and La3+ inhibited the LCC. Single-cell RT-PCR analysis revealed that mRNA of PKD2 and PKD2L2, but not PKDL or PKD1 are expressed in individual rat left ventricular myocytes. Conclusion: The characteristics of LCC shown in the present study are nearly identical to those observed for polycystin-2 and its homologues suggesting that polycystin-2 or polycystin-2L2 underlie LCC in ventricular myocytes.

KEYWORDS Ca-channel; Calcium (cellular); Ion channels; Myocytes; Na channel; Single channel currents


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Large conductance nonselective cation channels (LCC) have been described in a number of preparations including the heart [1–4]. They have in common a relatively large single channel conductance (100–300 pS), are permeable for monovalent and divalent cations but impermeable for anions. In the heart, LCC have been described and characterized in atrial myoballs isolated from the guinea pig [3] and in ventricular myocytes isolated from rabbit heart [4]. In atrial myoballs, LCC displayed a single channel conductance of 280 pS (for monovalent cations) with several subconductance states and were sensitive to octanol. Based on these characteristics, it was suggested that the channels may be related to gap-junction hemichannels [3]. LCC in rabbit ventricular myocytes were found to be permeable for monovalent as well as divalent cations, the single channel conductance was ~380 pS (for monovalent cations) and was reduced by ruthenium red or ryanodine. It was therefore suggested that the channel may be related to the ryanodine receptor (RyR) channel [4]. However, several important questions remained unanswered by these studies: gap-junction hemichannels are usually closed under conditions under which LCC have been recorded and it is unclear why and how the RyR channel should be present in the sarcolemmal membrane of cardiac myocytes.

Recent studies suggest that a potential molecular candidate for LCC may be formed by polycystin-2 or its homologues polycystin-L and polycystin-2L2. Polycystins are proteins encoded by the polycystic kidney disease genes PKD1, PKD2, PKDL and PKD2L2, of which mutations in PKD1 or PKD2 underlie autosomal dominant polycystic kidney disease (ADPKD; for review, see Ref. [5]). Polycystin-2, polycystin-L, and polycystin-2L2 share structural similarities with Na+ and Ca2+ channels, and it has been shown recently that polycystin-2 and polycystin-L form ion channels which are permeable for monovalent and divalent cations with conductances in the range of 30–500 pS when investigated in heterologous expression systems [6,7]. Moreover, it has been shown that PKD2, PKDL, and PKD2L2 are expressed in the heart [8–10]. We therefore questioned whether channels formed by polycystin-2, polycystin-L, or polycystin-2L2 may underlie the LCC in the heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 Patch clamp technique
Experiments were carried out on single myocytes isolated from the left ventricular free wall of female (unless stated otherwise) Sprague–Dawley rats (150–300 g). Briefly, after induction of anesthesia by i.p. injection of Trapanal (thiopental-sodium, 100 mg/kg body mass), the heart was quickly excised and placed in cold (4°C) cardioplegic solution. The aorta was cannulated and retrogradely perfused for 5 min with nominally Ca2+-free modified Tyrode's solution, followed by 15 min of perfusion with the same solution containing collagenase (type CLS II, 200 U/ml, Biochrom KG, Berlin, Germany) and protease (type XIV, 0.7 U/ml, Sigma, St Louis, MO, USA). Finally, the heart was perfused for 5 min with modified Tyrode's solution containing 100 µM Ca2+. After the perfusion, tissue pieces from the left ventricular free wall were carefully removed using fine forceps, further disaggregated by panning in modified Tyrode's solution containing 100 µM Ca2+ at 37°C, and then filtered through a cotton mesh. Isolated myocytes were stored in modified Tyrode's solution containing 100 µM Ca2+.

The ruptured-patch whole-cell configuration was used as described [11,12]. Membrane currents were recorded using an EPC-9 (Heka-Elektronik, Lambrecht, Germany) amplifier controlled by a Power-Macintosh computer (Apple Computer Inc., CA, USA) using the Pulse software (Heka-Elektronik). Pipette resistance averaged 2.4±0.03 M{Omega} (n = 709) with CsCl in the pipette, and 3.7±0.2 M{Omega} (n = 20) with K-glutamate in the pipette and control-solution in the bath. The series resistance (5.5±0.1 M{Omega}, n = 716) was compensated by 85%. Accordingly, at the largest recorded currents of about 1 nA, the voltage error was less than 1 mV. VPip and Vm were corrected for liquid junction potentials. Whole-cell currents were low-pass filtered at 1 kHz and sampled at 5 kHz. Single channel current traces were filtered off-line with 50–100 Hz using the Gaussian procedure of the Patch-program (Dr Bernd Letz). Channel open probability (NPo) was calculated by dividing the integral of the current trace above the current level at which all channels are closed by the single channel current amplitude and by the time of analysis using the Patch-program. Data are given as mean±S.E.M., statistical significance was calculated by the appropriate version of Student's t-test. Differences with P<0.05 were considered significant. Experiments were carried out at room temperature (20–24°C) if not stated otherwise. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by local authorities.

2.2 Solutions and chemicals
Cardioplegic solution contained (in mM) NaCl 15, KCl 9, MgCl2 4, NaH2PO4 0.33, CaCl2 0.015, glucose 10, mannitol 238, titrated to pH 7.40 with NaOH. Giga-ohm seals were obtained in modified Tyrode's solution (control solution, in mM): NaCl 142, MgCl2 1, NaH2PO4 0.33, CaCl2 1, glucose 10, Hepes 10, titrated to pH 7.30 with NaOH. In some experiments, 4 mM 4-aminopyridine and 0.1 mM BaCl2 were included to inhibit K+ currents. To evaluate the permeability for monovalent cations, the bath solution was similar to control solution except that it contained 140 mM NaCl and was titrated to pH 7.30 using Tris. From this solution, either 70 or 140 mM NaCl were substituted by an equal amount of either KCl, LiCl, CsCl, or N-methyl-D-glucamin-Cl (NMDG-Cl). Permeability for divalent cations was estimated using the following bath solutions (in mM): NaCl 97, sucrose 100, glucose 10, Hepes 10, titrated to pH 7.30 with Tris. NaCl and sucrose were substituted by either 97 CaCl2, 97 BaCl2 or 97 MgCl2. The pipette solution contained (in mM) CsCl 140, MgCl2 5, EGTA 0.1, Hepes 10, Na2ATP 2, titrated to pH 7.20 with CsOH (140CsCl solution). In some experiments, 20 mM CsCl was replaced by 20 mM tetraethylammonium-Cl to inhibit K+ currents (120CsCl solution). When indicated, the pipette solution contained 10 mM EGTA instead of 0.1 mM to reduce the intracellular concentration of free Ca2+. In some experiments, a Cs3-citrate-based pipette solution was used (in mM): citric acid 65, CsCl 10, MgCl2 5, NaCl 2, Hepes 10, Na2ATP 4, pH 7.20 with CsOH. For experiments using the perforated-patch configuration, the pipette solution contained K-glutamate 110, KCl 10, NaCl 10, MgCl2 1, CaCl2 1, Hepes 5, amphotericin B 250 µM, titrated to pH 7.20 with KOH.

2.3 Single-cell RT-PCR
cDNA synthesis and single-cell RT-PCR were carried out as described previously [13]. Briefly, under visualization a single myocyte was sucked into a micropipette and transferred into a reaction cup in which, after short centrifugation and a freeze–thaw cycle, reverse transcription using gene-specific primers followed. Subsequently, two consecutive PCRs with heminested primer pairs were carried out. Primer pairs were intron-overspanning to identify a possible amplification of genomic DNA. PCR products were identified by sequencing. Positive controls for primer efficiency were run using plasmids at several dilutions (down to 0.1 fg plasmid DNA). PCR primer sequences for rat PKD1, PKD2, PKDL, and PKD2L2 were: PKD1 upper primer: 5'-GGAGCGCTGGCCGGAGACCCTGG-3'; PKD1 lower primer: 5'-TGGAGAGGCAGGAAAGGTGTG-3'. PKD1 upper nested primer: 5'-CGAGTCTGCGCATCCCGGCTGA-3'. PKD2 upper primer: 5'-GGGACCCGCTGCATCGCCACC-3'; PKD2 lower primer: 5'-CTCATAGAAAATAAAGCTCCGGTTGTCAG-3'. PKD2 upper nested primer: 5'-CCGAGAGGCTGGTGCGAGGAC-3'. PKDL upper primer: 5'-GGCAGGCTCACAAGCTACAG-3'; PKDL lower primer: 5'-CTCTCCCATCAGTCGGTTCAC-3'. PKDL upper nested primer: 5'-TTCAGGATCCAGACAAGCCAG-3'. PKD2L2 upper primer: 5'-GTCGTCCACGCTATCCCGCTG-3'; PKD2L2 lower primer: 5'-CAACACAGGAACCAGCTATGACC-3'. PKD2L2 upper nested primer: 5'-AGCTTCGCCATCATGTTCTTC-3'. RT primer sequences were: PKD2: 5'-CGGCACTCCTAGCAGCAG-3'; PKDL: 5'-ACGTGTCTGGCTGCTGTAGG-3'; PKD2L2: 5'-GTTGTGTGAAATTTGTGAGCG-3'.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Large conductance cation channels in left ventricular myocytes
Ion channels formed by polycystin-2 and polycystin-L have been shown to be activated by an increase in the intracellular Ca2+ concentration ([Ca2+]i). In the present study, we used caffeine to transiently increase [Ca2+]i. Fig. 1 shows the effects of caffeine on membrane currents recorded from three different myocytes (A, B, C) at a continuous holding potential of VPip=–90 mV. In Fig. 1A, the transient activation of an inward current is recorded after a delay of ~5 s following the switch to caffeine-containing bath solution. This transient inward current was not recorded in the presence of 5 mM Ni2+ in the bath solution (data not shown) or when internal Ca2+ was buffered with 10 mM EGTA (Fig. 2A), and has previously been identified as the Na+/Ca2+ exchanger current (INa/Ca) [14,15]. Fig. 1B shows a similar recording in which on top of INa/Ca a single channel with a current amplitude of ~30 pA is activated, whereas in another recording (Fig. 1C), activation of at least three distinct single channels with identical current amplitude can be distinguished. Channel open probability (NPo) reached a maximum shortly after activation of INa/Ca, then declined and eventually reached a steady-state or decreased to zero. The inset displays the frequency of observation of the number of individual channels in single cells. On average, activation of single channel transitions of this LCC was observed in 56% of myocytes investigated (total n = 651); in 21% >1 individual channels were identified. Similar results were also obtained in myocytes isolated from male Sprague–Dawley rats. In 50% of the myocytes (total n = 30), one or more LCC were present upon application of caffeine to the bath solution (data not shown).


Figure 1
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  		Fig. 1 (A–C) Effect of caffeine (10 mM) on whole cell currents recorded from three different myocytes. The pipette solution contained 120CsCl solution with 0.1 mM EGTA. The bath solution contained control solution with 4 mM 4-AP and 0.1 mM BaCl2. In (A) a transient inward current is observed which is carried by forward-mode INa/Ca (see text). In (B) and (C), single channel transitions with an amplitude of ~30 pA were observed in addition to INa/Ca. The inset indicates the frequency of occurrence of the number of individual channels (from a total of 651 recordings).

 

Figure 2
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  		Fig. 2 (A) Similar recordings as in Fig. 1, except that the pipette solution contained 10 mM instead of 0.1 mM EGTA. The horizontal bar indicates the presence of caffeine in the bath solution. (B) Effect of depolarization-induced Ca2+-influx on membrane currents at VPip=–90 mV in the absence of caffeine. VPip was clamped for 250 ms to 0 mV and then back to VPip=–90 mV. The pipette solution contained 65 mM Cs3-citrate (see Methods). The bath solution contained control solution.

 
Caffeine opens the RyR channel and thus quickly releases Ca2+ from the SR [16], which increases [Ca2+]i approximately up to 1 µM in rat ventricular myocytes [15]. The close association of INa/Ca and the activation of LCC together with the consecutive decrease in NPo after cessation of INa/Ca suggest that an increase in [Ca2+]i is responsible for activation of the channel. To further test this hypothesis, we carried out experiments, in which intracellular Ca2+ was buffered using 10 mM EGTA in the pipette solution (n = 29). Fig. 2A shows a representative recording: upon application of caffeine, no INa/Ca can be observed and only rare openings of LCC are noted. On average, NPo (calculated over a period of 30 s after application of caffeine in those recordings, in which channel activity was noted) was 30 times lower in the presence of 10 mM EGTA in the pipette solution than in its absence (0.009±0.003, n = 13 vs. 0.310±0.06 n = 39, P<0.001), suggesting that Ca2+ contributes to channel activation. Furthermore, an increase in extracellular [Ca2+] from 1 to 10 mM resulted in an increase in NPo of LCC by a factor of 17±5 (n = 19, P<0.05).

The presence of single channel activity upon application of caffeine in the absence of an increase in [Ca2+]i could suggest that caffeine may be required to activate the LCC. This is further supported by the observation that a sustained activity of LCC was noted in the presence of caffeine in the bath solution which always ceased after removal of caffeine. To test whether LCC can also be activated by an increase in [Ca2+]i in the absence of caffeine, a different voltage-pulse protocol was used. Myocytes were stepped for 250 ms to VPip=0 mV to activate depolarisation-induced Ca2+-influx/SR-release and then back to VPip=–90 mV to identify LCC. The pipette contained 65 mM Cs3-citrate, which is known to decelerate the inactivation of the L-type Ca2+ current and to induce regenerative Ca2+-release from the SR thus leading to larger intracellular Ca2+ transients [17]. Fig. 2B displays a representative recording. Upon returning VPip back to –90 mV after the depolarisation, activation of a LCC can clearly be identified. Using this approach, LCC were detected in 30% of all cells investigated (n = 10). These results indicate that activation of LCC do not require the presence of caffeine.

To address the possibility that LCC are artificially formed during the generation of the whole-cell configuration as a consequence of the rupture of the plasma membrane, we carried out similar experiments using the perforated patch technique. Application of caffeine (10 mM) resulted in activation of the LCC in seven of eight attempts (data not shown) indicating that mechanical rupture of the plasma membrane is not responsible for LCC formation or activation.

3.2 Biophysical properties of the LCC
In recordings in which LCC remained active for a prolonged period after cessation of INa/Ca, channel properties were further investigated. Fig. 3A shows current traces with single channel transitions recorded from one myocyte at holding potentials between VPip=–80 and –10 mV. Single channel amplitude progressively decreased with increasing VPip. Myocytes could only be clamped to positive membrane potentials for a short period (<1 s), since they hypercontracted upon the depolarization resulting in a loss of the recording. However, in experiments in which internal Ca2+ was buffered using 10 mM EGTA, myocytes could be clamped to positive membrane potentials and outward single channel transitions were recorded from VPip=+30 to +90 mV. The average current–voltage (I–V) relation is shown in Fig. 3B. The outward conductance was smaller than the inward conductance, suggesting that the channel is either less permeable for Cs+ than for Na+, or is selective for Cl– with an inwardly rectifying characteristic.


Figure 3
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  		Fig. 3 (A) Single channel transitions recorded at holding potentials ranging from VPip=–80 to –10 mV in a single left ventricular myocyte. (B) Average I–V relation derived from 31 experiments. Outward transitions were recorded in experiments, in which the pipette solution contained 10 mM EGTA. (C) Current trace demonstrating the simultaneous presence of two conductance levels. The solid line indicates the current level at which all channels are closed, the dotted lines indicate the open states of the larger conductance level at a current amplitude of 30 pA. The pipette solution contained 140CsCl solution with 0.1 mM EGTA, the bath contained control solution and 10 mM caffeine.

 
Although the most often observed transition level was ~300 pS, smaller conductance levels with ~150 or ~75 pS were also occasionally noted. Fig. 3C shows a current recording in which the simultaneous occurrence of two conductance levels (~300 and ~150 pS) was noted. In five experiments, the single channel amplitude was estimated at 37°C and averaged –42±2 pA (VPip=–90 mV). The resulting Q10 was ~1.4, a value that has been found for many nonselective cation channels (NSC) [18].

Fig. 4A shows current traces recorded at VPip=–90 mV in the presence of 140, 70 or 0 mM NaCl in the bath. Single channel amplitude decreased with 70 mM NaCl in the bath and when extracellular Na+ was completely replaced by NMDG+, which was only possible for brief periods since the myocytes contracted due to Ca2+ entry via the reverse mode of the Na+/Ca2+ exchanger, single channel transitions became undetectable. This demonstrates that the LCC is permeable for Na+, but not for Cl–. The corresponding average I–V relations are shown in Fig. 4B. With 140 mM Na+ in the bath (filled symbols), the average I–V from 24 similar recordings revealed an average single channel conductance of 307 pS. Exchange of 70 mM of extracellular NaCl by the large organic cation NMDG-Cl reduced the inward single channel amplitude to about 50%. Both I–V values were well fitted using the Goldmann-Hodgkin-Katz equation with the assumption that the channel is selective for monovalent cations. To determine the permeability of LCC for other monovalent cations, single channel amplitude was recorded at VPip=–90 mV with 70 mM extracellular Na+ replaced by an equimolar amount of either NMDG+, K+, Cs+ or Li+ (Fig. 4C). The LCC is equally permeable for Na+ and K+, but slightly less for Cs+ and Li+. Taken together, the LCC is selective for cations over chloride, but does only weakly discriminate between monovalent cations.


Figure 4
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  		Fig. 4 (A) Single channel current traces recorded at VPip=–90 mV with 140 mM Na+ (upper trace), 70 mM Na+ (middle trace), or 0 mM Na+ (lower trace) in the bath solution. (B) Corresponding average I–V relations recorded in the presence of 140 mM NaCl in the bath (filled symbols, n = 24) or after replacement of 70 mM NaCl by an equimolar amount of NMDG-Cl (open symbols, n = 4). I–V relations were fitted using the Goldmann-Hodgkin-Katz equation with the assumption of a selective monovalent permeability (see Results). (C) Single channel current amplitudes recorded at VPip=–90 mV in the presence of 140 mM NaCl in the bath or after replacement of 70 mM NaCl by an equal amount of either NMDG-Cl, KCl, CsCl, or LiCl. ***P<0.001 versus 140 mM NaCl, n.s., not significant. The pipette contained 140CsCl solution with 0.1 mM EGTA in all experiments.

 
The permeability of LCC for divalent cations was investigated by increasing extracellular Ba2+, Ca2+ or Mg2+ to 97 mM. All monovalent cations were removed from the bath solution and osmolality was maintained by addition of sucrose (100 mM). With 97 mM Ba2+ in the bath solution, single channel amplitude averaged 20.2±1.1 pA (n = 8) at VPip=–90 mV, whereas with 97 mM Na+, it averaged 23.0±0.6 pA (n = 8). To avoid contraction of myocytes, intracellular Ca2+ was buffered using 10 mM EGTA in the pipette solution when the bath solution was switched to 97 mM Ca2+. Under these conditions, single channel amplitude averaged 17.2±0.7 pA (n = 3) at VPip=–90 mV. In contrast, when the bath solution was switched from 97 mM Na+ to 97 mM Mg2+, transitions disappeared (n = 6). These results demonstrate that the LCC is permeable for Ba2+ and Ca2+, but not for Mg2+.

To investigate the effects of divalent cations on the permeability of the LCC for monovalent cations, extracellular Mg2+, Ca2+, or Ba2+ was increased to 10 mM. Fig. 5 depicts the effect of 10 mM Mg2+ on the single channel conductance recorded at VPip=–90 mV. In this experiment, the initial single channel amplitude was 29 pA, but when the Mg2+ concentration was increased from 1 to 10 mM, single channel amplitude decreased by almost 50% to 17 pA. On average, single channel amplitude decreased from a control value of 28.9±0.4 to 17.8±0.7 pA (n = 8; P<0.0001). Similar results were obtained for 10 mM Ba2+ (19.1±1.2 vs. 28.3±0.4 pA; n = 5; P<0.01) and 10 mM Ca2+ (19.8±1.1 vs. 27.5±0.9 pA; n = 12; P<0.0001). A modulation of monovalent cation permeability by divalent cations has been reported previously for polycystin-2 and polycystin-L channels [7,19].


Figure 5
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  		Fig. 5 Single channel transitions of LCC recorded at VPip=–90 mV in the presence of 1 mM (A) and 10 mM (B) extracellular Mg2+. The pipette contained 140CsCl solution with 0.1 mM EGTA. The bath contained control solution. Amplitude histograms were calculated over the whole current recording depicted above. The single channel amplitude was estimated from the amplitude histograms as well as by fitting equidistant current levels to the current recordings. Both techniques yielded the same current amplitude, indicated below the current traces.

 
3.3 Pharmacology of the LCC
To further characterize the LCC, the effects of several substances known to affect polycystin channels and other NSC were investigated. Fig. 6A shows the effect of 500 µM amiloride on the LCC in a single recording. In this experiment, amiloride reversibly reduced NPo by 72%. The inset displays a dose–response relation of the effect of amiloride on LCC, fitted by the Hill equation. Half-maximal inhibition was observed at 131±1.1 µM, which is similar to that observed for polycystin-2 (27–79 µM) [6]. Gd3+ and La3+, which inhibit stretch activated NSCs in micromolar concentrations [20], and polycystin channels and other NSC in the millimolar range [6,7,19,21], had no effect on NPo or single channel conductance of the LCC at 0.1 mM (n = 4 for Gd3+, n = 10 for La3+), but irreversibly reduced NPo to zero at a concentration of 1 mM (n = 6 for Gd3+, n = 8 for La3+). Fig. 6B shows the effect of 5 mM Ni2+ on LCC activity. Ni2+ reversibly reduced NPo by an average of 73±9% (n = 12).


Figure 6
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  		Fig. 6 (A) Effect of amiloride (500 µM) on LCC. On average, NPo decreased by 97±3% (n = 9). The inset displays a concentration–response relation of amiloride, half-maximal inhibition was observed at 131±1.1 µM. (B) Effect of Ni2+ (5 mM) on LCC. (C) Effect of 18-{alpha}-glycyrrhetinic acid (20 µM) on LCC. (D) LCC activity recorded after application of caffeine (10 mM) in the presence of 25 µM ryanodine in the pipette solution. In this recording, caffeine was applied 4 min after breakthrough into the whole-cell configuration to allow for a sufficient diffusion time for the ryanodine. The single channel amplitude was calculated using the amplitude histogram shown on the right and averaged 30 pA.

 
It has been suggested that a LCC observed in guinea pig atrial myoballs may be related to gap junction hemichannels [3]. We therefore tested the effect of the gap junction hemichannel inhibitor 18-{alpha}-glycyrrhetinic acid (20 µM) [22]. 18-{alpha}-glycyrrhetinic acid had no effect on either NPo or single channel conductance (Fig. 6C, n = 11). Ruthenium red, an inhibitor of RyR channels (and also of other cation permeable channels [23]), reduced the single channel amplitude of a LCC in rabbit ventricular myocytes [4]. We therefore investigated the effects of ruthenium red (10 µM) in the bath solution. In 15 of 16 experiments, ruthenium red had no effect on single channel conductance. In one experiment, we observed a shift to a lower conductance state. Ryanodine has also been reported to reduce the single channel amplitude of a LCC in rabbit ventricular myocytes when applied intra- or extracellularly [4]. In our hands, ryanodine (10 µM) in the bath solution had no effect on NPo or single channel conductance (n = 13). Pre-incubation of myocytes with ryanodine for up to 3 min (n = 8) did not prevent the LCC from activation nor altered its NPo or single channel conductance upon application of caffeine. Also, in the presence of ryanodine (25 µM) in the pipette solution, the single channel amplitude was similar to that observed in the absence of ryanodine (Fig. 6D, n = 32). On no occasion did we observe a shift to a lower conductance level during the course of the experiments. Application of caffeine after prolonged incubation of myocytes with ryanodine resulted in a reduced amplitude or complete absence of contraction and INa/Ca. This confirms the intracellular presence of ryanodine and its action on the RyR channels. Taken together, these results argue against the hypothesis that the LCC in rat ventricular myocytes is related to gap junction hemichannels or the RyR.

3.4 Expression of PKD1, PKD2, PKDL, and PKD2L2 in ventricular myocytes
Expression of mRNA of PKD1, PKD2 and its homologues PDKL and PKD2L2 has been demonstrated in murine and human whole heart preparations [9,10,24]. To confirm the presence of all four mRNAs in the rat heart, RT-PCR with primers specific for each gene was carried out on total RNA isolated from the rat left ventricle. Fig. 7A shows an agarose gel with PCR products of the expected length for PKD2, PDKL, and PKD2L2, and Fig. 7B an agarose gel with a PCR product of the expected length for PKD1. All PCR products were identified by sequencing. These experiments show that PKD1, PKD2 and its homologues are expressed in the rat left ventricle. They do not exclude, however, the possibility that PKD1, PKD2, PDKL, and PKD2L2 may only be expressed in non-myocyte tissue such as blood vessels, connective tissue, or cardiac nerves. To test the expression of PKD1, PKD2, PKDL, and PKD2L2 mRNA in cardiac myocytes directly, single-cell RT-PCR was carried out on isolated left ventricular myocytes. Fig. 7C+D shows representative results obtained by single-cell RT-PCR for PKD2 (Fig. 7C) and PKD2L2 (Fig. 7D). In total, PKD2 was detected in seven of 20 (35%) myocytes and PKD2L2 in six of 20 (30%) myocytes. In contrast, neither PKDL (n = 20) nor PKD1 (n = 70) were detected in any myocytes investigated. Similar results were obtained when the identical RT-PCR protocol was carried out on pools of 5–10 myocytes that were consecutively sucked into a micropipette (n = 5). The absence of PKDL mRNA from myocytes is consistent with a recent study which detected polycystin-L in the epicardium and in ventricular blood vessels, but not in cardiac myocytes of the mouse [25]. Taken together, these data show that left ventricular myocytes express PKD2 and PKD2L2, whereas PKDL and PKD1 expression appear to be limited to non-myocyte cardiac tissue.


Figure 7
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  		Fig. 7 (A,B) Ethidium bromide-stained gels of RT-PCR products, amplified from total cardiac RNA with primers specific for PKD2, PKDL, and PKD2L2 (A) and for PKD1 (B). (C,D) Ethidium bromide-stained gel of single-cell RT-PCR products, amplified from individual left ventricular myocytes (1–5) with primers specific for PKD2 (C) and PKD2L2 (D). C1, C2, controls for each round of RT-PCR; RT, control for reverse transcription; TC, control for surrounding tissue; M, molecular weight marker; 200 and 500 refer to the corresponding number of base pairs.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 References
 
4.1 Mechanism of activation
The close association of LCC activation with INa/Ca upon application of caffeine to the bath solution suggests that an increase in [Ca2+]i at least participates in activation of LCC. This is supported by the observation that channel NPo was much lower when caffeine was applied in the presence of EGTA in the pipette solution. Furthermore, using a citrate-based pipette solution, depolarization induced Ca2+-influx/SR-release transiently activated LCC in the absence of caffeine thus demonstrating that an increase in [Ca2+]i alone is sufficient to activate LCC. Similar results have been obtained in guinea pig atrial myoballs: cyclic increases in [Ca2+]i observed with a citrate-based pipette solution activated LCC in the absence of caffeine [3]. On the other hand, we consistently observed LCC activity, although with very low open probability, upon application of caffeine in the presence of EGTA in the pipette solution, suggesting that caffeine alone (or together with very low levels of [Ca2+]i) can activate LCC. Similar results have been observed for LCC in rabbit ventricular myocytes [4]. It therefore appears likely that application of caffeine to the bath solution activates LCC by both an increase in [Ca2+]i and by a direct effect of caffeine on the channel.

4.2 Relation of the LCC to other cardiac nonselective cation channels
In excised inside-out patches from isolated ventricular myocytes, Ca2+-activated NSC with a single channel conductance of ~20 pS have been described [26]. Similar NSC have been detected in a variety of tissues (for review, see Ref. [18]). A potential molecular basis of these channels may be the large family of transient receptor potential (TRP) channels since this channel family comprises members which are Ca2+ permeable NSCs (e.g. TRP1, TRP3 and TRP6) [27]. TRP channels are found in many tissues including the heart, but they differ from the LCC described in the present investigation with respect to their single channel conductance (which is considerably lower), and pharmacology [28], thus making it unlikely that the LCC belongs to the TRP channel family.

The rat LCC characterized in the present study shares many properties with two LCC which were previously described in the hearts of guinea pigs and rabbits [3,4]. These channels show a similar single channel conductance, permeability for monovalent and divalent cations, and regulation by [Ca2+]. The LCC described in rabbit ventricular myocytes was inhibited by ruthenium red and ryanodine, and it was suggested that this LCC may be related to the RyR channel [4]. Despite several similarities, the LCC investigated in the present study was not affected either by extra- or by intracellularly applied ryanodine, even though we used identical concentrations of ryanodine and its intracellular action was apparent by the absence of SR Ca2+ release upon caffeine application after prolonged incubation with ryanodine. Thus LCC in rat ventricular myocytes appears to be similar, but not identical to LCC described in cardiac myocytes of guinea pigs and rabbits.

4.3 Relation of the LCC to ion channels formed by polycystins
PKD1 [29], PKD2 [24], PKDL [9], and PKD2L2 [10] are expressed in the mouse and human heart, but it was unclear whether this expression actually occurs in cardiomyocytes. Using single-cell RT-PCR, we could demonstrate that in the rat heart, PKD2 and PKD2L2 are expressed in left ventricular myocytes, while the expression of PKD1 and PKDL appears to be limited to non-myocyte tissue. Previous studies have shown that the expression of channel mRNA correlates with the expression of the corresponding ion channel proteins in cardiac as well as non-cardiac tissue [30,31]. It therefore seems likely that polycystin-2 and polycystin-2L2 are also expressed in rat left ventricular myocytes. In human embryonic hearts, polycystin-2 was detected in myocytes and in the endocardium [32]. In contrast, a recent study investigating the expression of polycystin-2 in the rat heart failed to detect a significant protein level, which, according to the authors, might have resulted from a low sensitivity of the antibody or from the preparation technique [33].

An important question is whether polycystin-2 and related proteins are actually located in the plasma cell membrane, or rather in intracellular membranes such as the endoplasmic reticulum. Recent studies suggested that polycystin-2 may be primarily located in intracellular membranes [34,35]. These results are supported by the observation that a nonselective cation conductance could only be observed when polycystin-2 was expressed together with polycystin-1 [19], or when the protein transport from intracellular compartments to the plasma membrane was enhanced [34]. In contrast, expression of polycystin-L in Xenopus oocytes alone produced nonselective cation currents [7] as did polycystin-2 when expressed in Sf9 cells [6]. These studies, together with our results, suggest that PKD2 and PKD2L2 are expressed in left ventricular myocytes and form functional ion channels in the plasma membrane. The relatively low abundance of channels that we have observed in the plasma membrane, may be explained by a lack of PKD1 expression in cardiac myocytes. Alternatively, the low number of LCC observed in single myocytes may actually result from a misdirected protein transport, either during the process of protein synthesis or as a consequence of the isolation procedure.

The LCC described in the present study displays biophysical and pharmacological properties very similar to channels formed by polycystin-2 and its homologues. All of them are permeable for mono- and divalent cations, but impermeable for anions. The single channel conductance of polycystin channels [6,7] is well within the range we have observed for the LCC. Both, LCC and polycystin channels, are activated by an increase in [Ca2+]i and inhibited by the trivalent cations Gd3+ and La3+ as well as amiloride at equal concentrations [6,7,19]. Furthermore, both LCC in the present study and polycystin-2 channels are insensitive to ryanodine [34]. Additional experiments with more specific inhibitors of polycystin channels (which are currently unavailable), or overexpressing polycystin-2 or polycystin-2L2 in cardiac myocytes are requested to further investigate the role of polycystins in cardiac myocytes.

4.4 Possible function of polycystins and LCC in the heart
The physiological role of polycystins in cardiac myocytes is still unclear. Patients with ADPKD are more likely to suffer from cardiac abnormalities such as septal deformations or mitral valve prolapse [8], suggesting a potential role of polycystins in the development of the heart. This notion is supported by a recent finding that mice with homozygous deletions of PKD2 die in utero and display severe cardiac abnormalities, particularly septal defects [36]. Moreover, expression levels of polycystin-2 are higher during embryonic development of the heart and decrease to lower levels at later stages of development [24].

It has been suggested that polycystin-2 participates in signal transduction pathways by modulating [Ca2+]i, possibly as a release channel [35,37]. Independent of the channel location, Ca2+-induced Ca2+ release is a tightly controlled mechanism in cardiac myocytes, and it is not quite clear which role an additional large NSC should play. However, given the relatively long periods during which openings of the LCC can be observed after an increase in [Ca2+]i, it seems possible that LCC may influence baseline [Ca2+]i and thus participate in Ca2+-mediated signal transduction pathways, such as signals that promote cardiac hypertrophy. In this respect, it is interesting to note that patients suffering from ADPKD develop diastolic dysfunction early in life, independent of additional risk factors like hypertension [38,39].


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
References
 
The present study shows that the LCC observed in rat left ventricular myocytes display biophysical properties, pharmacological profile, and regulation by [Ca2+]i which are very similar to channels formed by polycystin-2, polycystin-L, and polycystin-2L2. Furthermore, single-cell RT-PCR analysis revealed that mRNAs of polycystin-2 and polycystin-2L2, but not polycystin-1 or polycystin-L are expressed in individual myocytes of the rat left ventricle. We therefore suggest that polycystin-2 or its homologues underlie LCC in rat left ventricular myocytes.

Time for primary review 22 days.


   Acknowledgements
 
We are most grateful to Telse Kock for expert technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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