Cardiovascular Research 58 (2003) 76–88 www.elsevier.com/locate/cardiores

A polycystin-2-like large conductance cation channel in rat left ventricular myocytes Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021 Tilmann Volk* , Alexander Peter Schwoerer, Susanne Thiessen, Jobst-Hendrik Schultz, Heimo Ehmke* Institut f ur¨¨ Physiologie, Universitatskrankenhaus Hamburg-Eppendorf, Universitat ¨ Hamburg, Martinistraße 52, 20246 Hamburg, Germany Received 2 July 2002; accepted 17 December 2002

Abstract Objective: Several members of the PKD family (PKD2, PKDL and PKD2L2 ) are expressed in the heart. Polycystin-2 and its homologues, which are encoded by these , have recently been shown to form Ca21 -regulated nonselective cation channels in heterologous expression systems. Previously, large conductance nonselective cation channels (LCC) have been described in car- diomyocytes, 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 Ca21 concentration led to activation of LCC in 56% of the myocytes investigated (total n5651), 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 Na11 , K , Li 1 , and Cs 1 and 21 2112 also permeable for the divalent cations Ca and Ba , but impermeable for NMDG and Cl . (IC50513161.1 mM) and millimolar concentrations of the trivalent cations Gd31 and La31 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.  2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.

Keywords: Ca-channel; (cellular); Ion channels; Myocytes; Na channel; Single channel currents

1 . Introduction with several subconductance states and were sensitive to octanol. Based on these characteristics, it was suggested Large conductance nonselective cation channels (LCC) that the channels may be related to gap-junction hemichan- have been described in a number of preparations including nels [3]. LCC in rabbit ventricular myocytes were found to the heart [1–4]. They have in common a relatively large be permeable for monovalent as well as divalent cations, single channel conductance (100–300 pS), are permeable the single channel conductance was |380 pS (for mono- for monovalent and divalent cations but impermeable for valent cations) and was reduced by red or anions. In the heart, LCC have been described and . It was therefore suggested that the channel may characterized in atrial myoballs isolated from the guinea be related to the (RyR) channel [4]. pig [3] and in ventricular myocytes isolated from rabbit However, several important questions remained unan- heart [4]. In atrial myoballs, LCC displayed a single swered by these studies: gap-junction hemichannels are channel conductance of 280 pS (for monovalent cations) 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 *Corresponding authors. Tel.: 149-40-42803-9615 (T. Volk); 149-40- 42803-3183 (H. Ehmke); fax: 149-40-42803-9299. cardiac myocytes. E-mail addresses: [email protected] (T. Volk), [email protected] (H. Ehmke). Time for primary review 22 days.

0008-6363/03/$ – see front matter  2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0008-6363(02)00858-1 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 77

Recent studies suggest that a potential molecular candi- tentials. Whole-cell currents were low-pass filtered at 1 date for LCC may be formed by polycystin-2 or its kHz and sampled at 5 kHz. Single channel current traces homologues polycystin-L and polycystin-2L2. Polycystins were filtered off-line with 50–100 Hz using the Gaussian are encoded by the polycystic kidney disease procedure of the Patch-program (Dr Bernd Letz). Channel genes PKD1, PKD2, PKDL and PKD2L2, of which open probability (NPo ) was calculated by dividing the mutations in PKD1 or PKD2 underlie autosomal dominant integral of the current trace above the current level at polycystic kidney disease (ADPKD; for review, see Ref. which all channels are closed by the single channel current [5]). Polycystin-2, polycystin-L, and polycystin-2L2 share amplitude and by the time of analysis using the Patch- structural similarities with Na1 and Ca21 channels, and it program. Data are given as mean6S.E.M., statistical has been shown recently that polycystin-2 and polycystin- significance was calculated by the appropriate version of L form ion channels which are permeable for monovalent Student’s t-test. Differences with P,0.05 were considered and divalent cations with conductances in the range of significant. Experiments were carried out at room tempera- Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021 30–500 pS when investigated in heterologous expression ture (20–24 8C) if not stated otherwise. The investigation systems [6,7]. Moreover, it has been shown that PKD2, conforms with the Guide for the Care and Use of Labora- PKDL, and PKD2L2 are expressed in the heart [8–10]. We tory Animals published by the US National Institutes of therefore questioned whether channels formed by Health (NIH Publication No. 85-23, revised 1996) and was polycystin-2, polycystin-L, or polycystin-2L2 may underlie approved by local authorities. the LCC in the heart. 2 .2. Solutions and chemicals

2 . Methods Cardioplegic solution contained (in mM) NaCl 15, KCl

9, MgCl2242 4, NaH PO 0.33, CaCl 0.015, 10, 2 .1. Patch clamp technique mannitol 238, titrated to pH 7.40 with NaOH. Giga-ohm seals were obtained in modified Tyrode’s solution (control

Experiments were carried out on single myocytes iso- solution, in mM): NaCl 142, MgCl224 1, NaH PO 0.33, lated from the left ventricular free wall of female (unless CaCl2 1, glucose 10, Hepes 10, titrated to pH 7.30 with stated otherwise) Sprague–Dawley rats (150–300 g). NaOH. In some experiments, 4 mM 4-aminopyridine and 1 Briefly, after induction of anesthesia by i.p. injection of 0.1 mM BaCl2 were included to inhibit K currents. To Trapanal (thiopental-sodium, 100 mg/kg body mass), the evaluate the permeability for monovalent cations, the bath heart was quickly excised and placed in cold (4 8C) solution was similar to control solution except that it cardioplegic solution. The aorta was cannulated and re- contained 140 mM NaCl and was titrated to pH 7.30 using trogradely perfused for 5 min with nominally Ca21 -free Tris. From this solution, either 70 or 140 mM NaCl were modified Tyrode’s solution, followed by 15 min of perfu- substituted by an equal amount of either KCl, LiCl, CsCl, sion with the same solution containing collagenase (type or N-methyl-D-glucamin-Cl (NMDG-Cl). Permeability for CLS II, 200 U/ml, Biochrom KG, Berlin, Germany) and divalent cations was estimated using the following bath protease (type XIV, 0.7 U/ml, Sigma, St Louis, MO, solutions (in mM): NaCl 97, 100, glucose 10, USA). Finally, the heart was perfused for 5 min with Hepes 10, titrated to pH 7.30 with Tris. NaCl and sucrose 21 modified Tyrode’s solution containing 100 mM Ca . were substituted by either 97 CaCl22 , 97 BaCl or 97 After the perfusion, tissue pieces from the left ventricular MgCl2 . The pipette solution contained (in mM) CsCl 140, free wall were carefully removed using fine forceps, MgCl22 5, EGTA 0.1, Hepes 10, Na ATP 2, titrated to pH further disaggregated by panning in modified Tyrode’s 7.20 with CsOH (140CsCl solution). In some experiments, solution containing 100 mMCa21 at378C, and then 20 mM CsCl was replaced by 20 mM tetraethylam- filtered through a cotton mesh. Isolated myocytes were monium-Cl to inhibit K1 currents (120CsCl solution). stored in modified Tyrode’s solution containing 100 mM When indicated, the pipette solution contained 10 mM Ca21 . EGTA instead of 0.1 mM to reduce the intracellular 21 The ruptured-patch whole-cell configuration was used as concentration of free Ca . In some experiments, a Cs3 - described [11,12]. Membrane currents were recorded using citrate-based pipette solution was used (in mM): citric acid an EPC-9 (Heka-Elektronik, Lambrecht, Germany) am- 65, CsCl 10, MgCl22 5, NaCl 2, Hepes 10, Na ATP 4, pH plifier controlled by a Power-Macintosh computer (Apple 7.20 with CsOH. For experiments using the perforated- Computer Inc., CA, USA) using the Pulse software (Heka- patch configuration, the pipette solution contained K-gluta-

Elektronik). Pipette resistance averaged 2.460.03 MV (n5 mate 110, KCl 10, NaCl 10, MgCl22 1, CaCl 1, Hepes 5, 709) with CsCl in the pipette, and 3.760.2 MV (n520) amphotericin B 250 mM, titrated to pH 7.20 with KOH. with K-glutamate in the pipette and control-solution in the bath. The series resistance (5.560.1 MV, n5716) was 2 .3. Single-cell RT-PCR compensated by 85%. Accordingly, at the largest recorded currents of about 1 nA, the voltage error was less than 1 cDNA synthesis and single-cell RT-PCR were carried mV. VPipand V m were corrected for liquid junction po- out as described previously [13]. Briefly, under visualiza- 78 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88

tion a single myocyte was sucked into a micropipette and maximum shortly after activation of INa/Ca, then declined transferred into a reaction cup in which, after short and eventually reached a steady-state or decreased to zero. centrifugation and a freeze–thaw cycle, reverse transcrip- The inset displays the frequency of observation of the tion using gene-specific primers followed. Subsequently, number of individual channels in single cells. On average, two consecutive PCRs with heminested primer pairs were activation of single channel transitions of this LCC was carried out. Primer pairs were intron-overspanning to observed in 56% of myocytes investigated (total n5651); identify a possible amplification of genomic DNA. PCR in 21% .1 individual channels were identified. Similar products were identified by sequencing. Positive controls results were also obtained in myocytes isolated from male for primer efficiency were run using plasmids at several Sprague–Dawley rats. In 50% of the myocytes (total dilutions (down to 0.1 fg plasmid DNA). PCR primer n530), one or more LCC were present upon application of sequences for rat PKD1, PKD2, PKDL, and PKD2L2 caffeine to the bath solution (data not shown). were: PKD1 upper primer: 59-GGAGCGCTGGCCGGAG- Caffeine opens the RyR channel and thus quickly Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021 21 21 ACCCTGG-39; PKD1 lower primer: 59-TGGAGAGGCA- releases Ca from the SR [16], which increases [Ca ]i GGAAAGGTGTG-39. PKD1 upper nested primer: 59-CG- approximately up to 1 mM in rat ventricular myocytes [15].

AGTCTGCGCATCCCGGCTGA-39. PKD2 upper primer: The close association of INa/Ca and the activation of LCC 59-GGGACCCGCTGCATCGCCACC-39; PKD2 lower together with the consecutive decrease in NPo after cessa- 21 primer: 59-CTCATAGAAAATAAAGCTCCGGTTGTC- tion of INa/Casuggest that an increase in [Ca ] i is AG-39. PKD2 upper nested primer: 59-CCGAGAGGCTG- responsible for activation of the channel. To further test GTGCGAGGAC-39. PKDL upper primer: 59-GGCAGGC- this hypothesis, we carried out experiments, in which TCACAAGCTACAG-39; PKDL lower primer: 59-CTCT- intracellular Ca21 was buffered using 10 mM EGTA in the CCCATCAGTCGGTTCAC-39. PKDL upper nested pipette solution (n529). Fig. 2A shows a representative primer: 59-TTCAGGATCCAGACAAGCCAG-39. PKD2L2 recording: upon application of caffeine, no INa/Ca can be upper primer: 59-GTCGTCCACGCTATCCCGCTG-39; observed and only rare openings of LCC are noted. On

PKD2L2 lower primer: 59-CAACACAGGAACCAGCTA- average, NPo (calculated over a period of 30 s after TGACC-39. PKD2L2 upper nested primer: 59-AGCTTCG- application of caffeine in those recordings, in which CCATCATGTTCTTC-39. RT primer sequences were: channel activity was noted) was 30 times lower in the PKD2: 59-CGGCACTCCTAGCAGCAG-39; PKDL: 59- presence of 10 mM EGTA in the pipette solution than in ACGTGTCTGGCTGCTGTAGG-39; PKD2L2: 59-GTTG- its absence (0.00960.003, n513 vs. 0.31060.06 n539, TGTGAAATTTGTGAGCG-39. P,0.001), suggesting that Ca21 contributes to channel activation. Furthermore, an increase in extracellular [Ca21 ]

from 1 to 10 mM resulted in an increase in NPo of LCC by a factor of 1765(n519, P,0.05). 3 . Results The presence of single channel activity upon application 21 of caffeine in the absence of an increase in [Ca ]i could 3 .1. Large conductance cation channels in left suggest that caffeine may be required to activate the LCC. ventricular myocytes This is further supported by the observation that a sus- tained activity of LCC was noted in the presence of Ion channels formed by polycystin-2 and polycystin-L caffeine in the bath solution which always ceased after have been shown to be activated by an increase in the removal of caffeine. To test whether LCC can also be 21 21 21 intracellular Ca concentration ([Ca ]i ). In the present activated by an increase in [Ca ]i in the absence of 21 study, we used caffeine to transiently increase [Ca ]i . caffeine, a different voltage-pulse protocol was used.

Fig. 1 shows the effects of caffeine on membrane currents Myocytes were stepped for 250 ms to VPip50mVto recorded from three different myocytes (A, B, C) at a activate depolarisation-induced Ca21 -influx/SR-release continuous holding potential of VPip5290 mV. In Fig. 1A, and then back to VPip5290 mV to identify LCC. The the transient activation of an inward current is recorded pipette contained 65 mM Cs3 -citrate, which is known to after a delay of |5 s following the switch to caffeine- decelerate the inactivation of the L-type Ca21 current and containing bath solution. This transient inward current was to induce regenerative Ca21 -release from the SR thus not recorded in the presence of 5 mM Ni21 in the bath leading to larger intracellular Ca21 transients [17]. Fig. 2B 21 solution (data not shown) or when internal Ca was displays a representative recording. Upon returning VPip buffered with 10 mM EGTA (Fig. 2A), and has previously back to 290 mV after the depolarisation, activation of a been identified as the Na1 /Ca21 exchanger current LCC can clearly be identified. Using this approach, LCC

(INa/Ca) [14,15]. Fig. 1B shows a similar recording in were detected in 30% of all cells investigated (n510). which on top of INa/Ca a single channel with a current These results indicate that activation of LCC do not require amplitude of |30 pA is activated, whereas in another the presence of caffeine. recording (Fig. 1C), activation of at least three distinct To address the possibility that LCC are artificially single channels with identical current amplitude can be formed during the generation of the whole-cell configura- distinguished. Channel open probability (NPo ) reached a tion as a consequence of the rupture of the plasma T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 79 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

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).

membrane, we carried out similar experiments using the 3 .2. Biophysical properties of the LCC perforated patch technique. Application of caffeine (10 mM) resulted in activation of the LCC in seven of eight In recordings in which LCC remained active for a attempts (data not shown) indicating that mechanical prolonged period after cessation of INa/Ca, channel prop- rupture of the plasma membrane is not responsible for erties were further investigated. Fig. 3A shows current LCC formation or activation. traces with single channel transitions recorded from one 80 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

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 21 presence of caffeine in the bath solution. (B) Effect of depolarization-induced Ca -influx on membrane currents at VPip5290 mV in the absence of caffeine. VPip was clamped for 250 ms to 0 mV and then back to VPip5290 mV. The pipette solution contained 65 mM Cs3 -citrate (see Methods). The bath solution contained control solution.

myocyte at holding potentials between VPip5280 and 210 suggesting that the channel is either less permeable for mV. Single channel amplitude progressively decreased with Cs11 than for Na , or is selective for Cl 2 with an inwardly increasing VPip. Myocytes could only be clamped to rectifying characteristic. positive membrane potentials for a short period (,1 s), Although the most often observed transition level was since they hypercontracted upon the depolarization re- |300 pS, smaller conductance levels with |150 or |75 pS sulting in a loss of the recording. However, in experiments were also occasionally noted. Fig. 3C shows a current in which internal Ca21 was buffered using 10 mM EGTA, recording in which the simultaneous occurrence of two myocytes could be clamped to positive membrane po- conductance levels (|300 and |150 pS) was noted. In five tentials and outward single channel transitions were re- experiments, the single channel amplitude was estimated at corded from VPip5130 to 190 mV. The average current– 37 8C and averaged 24262pA(VPip5290 mV). The | voltage (I–V ) relation is shown in Fig. 3B. The outward resulting Q10 was 1.4, a value that has been found for conductance was smaller than the inward conductance, many nonselective cation channels (NSC) [18]. T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 81 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

Fig. 3. (A) Single channel transitions recorded at holding potentials ranging from VPip5280 to 210 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.

11 Fig. 4A shows current traces recorded at VPip5290 mV but slightly less for Cs and Li . Taken together, the LCC in the presence of 140, 70 or 0 mM NaCl in the bath. is selective for cations over chloride, but does only weakly Single channel amplitude decreased with 70 mM NaCl in discriminate between monovalent cations. the bath and when extracellular Na1 was completely The permeability of LCC for divalent cations was replaced by NMDG1 , which was only possible for brief investigated by increasing extracellular Ba21 , Ca21 or periods since the myocytes contracted due to Ca21 entry Mg21 to 97 mM. All monovalent cations were removed via the reverse mode of the Na1 /Ca21 exchanger, single from the bath solution and osmolality was maintained by channel transitions became undetectable. This demon- addition of sucrose (100 mM). With 97 mM Ba21 in the strates that the LCC is permeable for Na12 , but not for Cl . bath solution, single channel amplitude averaged 20.261.1 1 The corresponding average I–V relations are shown in Fig. pA (n58) at VPip5290 mV, whereas with 97 mM Na , it 4B. With 140 mM Na1 in the bath (filled symbols), the averaged 23.060.6 pA (n58). To avoid contraction of average I–V from 24 similar recordings revealed an myocytes, intracellular Ca21 was buffered using 10 mM average single channel conductance of 307 pS. Exchange EGTA in the pipette solution when the bath solution was of 70 mM of extracellular NaCl by the large organic cation switched to 97 mM Ca21 . Under these conditions, single

NMDG-Cl reduced the inward single channel amplitude to channel amplitude averaged 17.260.7 pA (n53) at VPip5 about 50%. Both I–V values were well fitted using the 290 mV. In contrast, when the bath solution was switched Goldmann-Hodgkin-Katz equation with the assumption from 97 mM Na1 to 97 mM Mg21 , transitions disappeared that the channel is selective for monovalent cations. To (n56). These results demonstrate that the LCC is perme- determine the permeability of LCC for other monovalent able for Ba21 and Ca21 , but not for Mg21 . cations, single channel amplitude was recorded at VPip52 To investigate the effects of divalent cations on the 90 mV with 70 mM extracellular Na1 replaced by an permeability of the LCC for monovalent cations, extracel- equimolar amount of either NMDG11 , K , Cs 1 or Li 1 lular Mg21 , Ca21 , or Ba21 was increased to 10 mM. Fig. 5 (Fig. 4C). The LCC is equally permeable for Na11 and K , depicts the effect of 10 mM Mg21 on the single channel 82 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

11 1 Fig. 4. (A) Single channel current traces recorded at VPip5290 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, n524) or after replacement of 70 mM NaCl by an equimolar amount of NMDG-Cl (open symbols, n54). 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 VPip5290 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.

conductance recorded at VPip5290 mV. In this experiment, mM amiloride on the LCC in a single recording. In this the initial single channel amplitude was 29 pA, but when experiment, amiloride reversibly reduced NPo by 72%. The the Mg21 concentration was increased from 1 to 10 mM, inset displays a dose–response relation of the effect of single channel amplitude decreased by almost 50% to 17 amiloride on LCC, fitted by the Hill equation. Half-maxi- pA. On average, single channel amplitude decreased from mal inhibition was observed at 13161.1 mM, which is a control value of 28.960.4 to 17.860.7 pA (n58; P, similar to that observed for polycystin-2 (27–79 mM) [6]. 0.0001). Similar results were obtained for 10 mM Ba21 Gd31 and La31 , which inhibit stretch activated NSCs in (19.161.2 vs. 28.360.4 pA; n55; P,0.01) and 10 mM micromolar concentrations [20], and polycystin channels Ca21 (19.861.1 vs. 27.560.9 pA; n512; P,0.0001). A and other NSC in the millimolar range [6,7,19,21], had no modulation of monovalent cation permeability by divalent effect on NPo or single channel conductance of the LCC at cations has been reported previously for polycystin-2 and 0.1 mM (n54 for Gd31 , n510 for La31 ), but irreversibly polycystin-L channels [7,19]. reduced NPo to zero at a concentration of 1 mM (n56 for Gd31 , n58 for La31 ). Fig. 6B shows the effect of 5 mM 21 21 3 .3. Pharmacology of the LCC Ni on LCC activity. Ni reversibly reduced NPo by an average of 7369% (n512). To further characterize the LCC, the effects of several It has been suggested that a LCC observed in guinea pig substances known to affect polycystin channels and other atrial myoballs may be related to hemichan- NSC were investigated. Fig. 6A shows the effect of 500 nels [3]. We therefore tested the effect of the gap junction T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 83 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

21 Fig. 5. Single channel transitions of LCC recorded at VPip5290 mV in the presence of 1 mM (A) and 10 mM (B) extracellular Mg . 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. hemichannel inhibitor 18-a-glycyrrhetinic acid (20 mM) This confirms the intracellular presence of ryanodine and

[22]. 18-a-glycyrrhetinic acid had no effect on either NPo its action on the RyR channels. Taken together, these or single channel conductance (Fig. 6C, n511). results argue against the hypothesis that the LCC in rat , an inhibitor of RyR channels (and also of ventricular myocytes is related to gap junction hemichan- other cation permeable channels [23]), reduced the single nels or the RyR. channel amplitude of a LCC in rabbit ventricular myocytes [4]. We therefore investigated the effects of ruthenium red 3 .4. Expression of PKD1, PKD2, PKDL, and PKD2L2 (10 mM) in the bath solution. In 15 of 16 experiments, in ventricular myocytes ruthenium red had no effect on single channel conduct- ance. In one experiment, we observed a shift to a lower Expression of mRNA of PKD1, PKD2 and its homo- conductance state. Ryanodine has also been reported to logues PDKL and PKD2L2 has been demonstrated in reduce the single channel amplitude of a LCC in rabbit murine and human whole heart preparations [9,10,24]. To ventricular myocytes when applied intra- or extracellularly confirm the presence of all four mRNAs in the rat heart, [4]. In our hands, ryanodine (10 mM) in the bath solution RT-PCR with primers specific for each gene was carried had no effect on NPo or single channel conductance (n5 out on total RNA isolated from the rat left ventricle. Fig. 13). Pre-incubation of myocytes with ryanodine for up to 3 7A shows an agarose gel with PCR products of the min (n58) did not prevent the LCC from activation nor expected length for PKD2, PDKL, and PKD2L2, and Fig. altered its NPo or single channel conductance upon appli- 7B an agarose gel with a PCR product of the expected cation of caffeine. Also, in the presence of ryanodine (25 length for PKD1. All PCR products were identified by mM) in the pipette solution, the single channel amplitude sequencing. These experiments show that PKD1, PKD2 was similar to that observed in the absence of ryanodine and its homologues are expressed in the rat left ventricle. (Fig. 6D, n532). On no occasion did we observe a shift to They do not exclude, however, the possibility that PKD1, a lower conductance level during the course of the PKD2, PDKL, and PKD2L2 may only be expressed in experiments. Application of caffeine after prolonged incu- non-myocyte tissue such as blood vessels, connective bation of myocytes with ryanodine resulted in a reduced tissue, or cardiac nerves. To test the expression of PKD1, amplitude or complete absence of contraction and INa/Ca. PKD2, PKDL, and PKD2L2 mRNA in cardiac myocytes 84 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021

Fig. 6. (A) Effect of amiloride (500 mM) on LCC. On average, NPo decreased by 9763% (n59). The inset displays a concentration–response relation of amiloride, half-maximal inhibition was observed at 13161.1 mM. (B) Effect of Ni21 (5 mM) on LCC. (C) Effect of 18-a-glycyrrhetinic acid (20 mM) on LCC. (D) LCC activity recorded after application of caffeine (10 mM) in the presence of 25 mM 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. directly, single-cell RT-PCR was carried out on isolated 7C) and PKD2L2 (Fig. 7D). In total, PKD2 was detected left ventricular myocytes. Fig. 7C1D shows representative in seven of 20 (35%) myocytes and PKD2L2 in six of 20 results obtained by single-cell RT-PCR for PKD2 (Fig. (30%) myocytes. In contrast, neither PKDL (n520) nor T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 85

4 . Discussion

4 .1. Mechanism of activation

The close association of LCC activation with INa/Ca upon application of caffeine to the bath solution suggests 21 that an increase in [Ca ]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 in- 21 duced Ca -influx/SR-release transiently activated LCC in Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021 the absence of caffeine thus demonstrating that an increase 21 in [Ca ]i alone is sufficient to activate LCC. Similar results have been obtained in guinea pig atrial myoballs: 21 cyclic increases in [Ca ]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 21 with very low levels of [Ca ]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 21 increase in [Ca ]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, Ca21 -activated NSC with a single channel conductance of |20 pS have been described [26]. Similar Fig. 7. (A,B) Ethidium bromide-stained gels of RT-PCR products, NSC have been detected in a variety of tissues (for review, amplified from total cardiac RNA with primers specific for PKD2, PKDL, see Ref. [18]). A potential molecular basis of these and PKD2L2 (A) and for PKD1 (B). (C,D) Ethidium bromide-stained gel of single-cell RT-PCR products, amplified from individual left ventricular channels may be the large family of transient receptor myocytes (1–5) with primers specific for PKD2 (C) and PKD2L2 (D). potential (TRP) channels since this channel family com- 21 C1, C2, controls for each round of RT-PCR; RT, control for reverse prises members which are Ca permeable NSCs (e.g. transcription; TC, control for surrounding tissue; M, molecular weight TRP1, TRP3 and TRP6) [27]. TRP channels are found in marker; 200 and 500 refer to the corresponding number of base pairs. 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 PKD1 (n570) were detected in any myocytes investigated. that the LCC belongs to the TRP channel family. Similar results were obtained when the identical RT-PCR The rat LCC characterized in the present study shares protocol was carried out on pools of 5–10 myocytes that many properties with two LCC which were previously were consecutively sucked into a micropipette (n55). The described in the hearts of guinea pigs and rabbits [3,4]. absence of PKDL mRNA from myocytes is consistent with These channels show a similar single channel conductance, a recent study which detected polycystin-L in the epi- permeability for monovalent and divalent cations, and cardium and in ventricular blood vessels, but not in cardiac regulation by [Ca21 ]. The LCC described in rabbit ven- myocytes of the mouse [25]. Taken together, these data tricular myocytes was inhibited by ruthenium red and show that left ventricular myocytes express PKD2 and ryanodine, and it was suggested that this LCC may be PKD2L2, whereas PKDL and PKD1 expression appear to related to the RyR channel [4]. Despite several similarities, be limited to non-myocyte cardiac tissue. the LCC investigated in the present study was not affected 86 T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 either by extra- or by intracellularly applied ryanodine, channels formed by polycystin-2 and its homologues. All even though we used identical concentrations of ryanodine of them are permeable for mono- and divalent cations, but and its intracellular action was apparent by the absence of impermeable for anions. The single channel conductance SR Ca21 release upon caffeine application after prolonged of polycystin channels [6,7] is well within the range we incubation with ryanodine. Thus LCC in rat ventricular have observed for the LCC. Both, LCC and polycystin 21 myocytes appears to be similar, but not identical to LCC channels, are activated by an increase in [Ca ]i and described in cardiac myocytes of guinea pigs and rabbits. inhibited by the trivalent cations Gd31 and La31 as well as amiloride at equal concentrations [6,7,19]. Furthermore, 4 .3. Relation of the LCC to ion channels formed by both LCC in the present study and polycystin-2 channels polycystins are insensitive to ryanodine [34]. Additional experiments with more specific inhibitors of polycystin channels (which

PKD1 [29], PKD2 [24], PKDL [9], and PKD2L2 [10] are currently unavailable), or overexpressing polycystin-2 Downloaded from https://academic.oup.com/cardiovascres/article/58/1/76/295654 by guest on 01 October 2021 are expressed in the mouse and human heart, but it was or polycystin-2L2 in cardiac myocytes are requested to unclear whether this expression actually occurs in car- further investigate the role of polycystins in cardiac diomyocytes. Using single-cell RT-PCR, we could demon- myocytes. strate that in the rat heart, PKD2 and PKD2L2 are expressed in left ventricular myocytes, while the expres- 4 .4. Possible function of polycystins and LCC in the sion of PKD1 and PKDL appears to be limited to non- heart myocyte tissue. Previous studies have shown that the expression of channel mRNA correlates with the expres- The physiological role of polycystins in cardiac sion of the corresponding proteins in cardiac myocytes is still unclear. Patients with ADPKD are more as well as non-cardiac tissue [30,31]. It therefore seems likely to suffer from cardiac abnormalities such as septal likely that polycystin-2 and polycystin-2L2 are also ex- deformations or mitral valve prolapse [8], suggesting a pressed in rat left ventricular myocytes. In human em- potential role of polycystins in the development of the bryonic hearts, polycystin-2 was detected in myocytes and heart. This notion is supported by a recent finding that in the endocardium [32]. In contrast, a recent study mice with homozygous deletions of PKD2 die in utero and investigating the expression of polycystin-2 in the rat heart display severe cardiac abnormalities, particularly septal failed to detect a significant level, which, according defects [36]. Moreover, expression levels of polycystin-2 to the authors, might have resulted from a low sensitivity are higher during embryonic development of the heart and of the antibody or from the preparation technique [33]. decrease to lower levels at later stages of development An important question is whether polycystin-2 and [24]. related proteins are actually located in the plasma cell It has been suggested that polycystin-2 participates in membrane, or rather in intracellular membranes such as the pathways by modulating [Ca21 ] , pos- endoplasmic reticulum. Recent studies suggested that i sibly as a release channel [35,37]. Independent of the polycystin-2 may be primarily located in intracellular channel location, Ca21 -induced Ca21 release is a tightly membranes [34,35]. These results are supported by the controlled mechanism in cardiac myocytes, and it is not observation that a nonselective cation conductance could quite clear which role an additional large NSC should play. only be observed when polycystin-2 was expressed to- However, given the relatively long periods during which gether with polycystin-1 [19], or when the protein trans- openings of the LCC can be observed after an increase in port from intracellular compartments to the plasma mem- [Ca21 ] , it seems possible that LCC may influence baseline brane was enhanced [34]. In contrast, expression of i [Ca21 ] and thus participate in Ca21 -mediated signal polycystin-L in Xenopus oocytes alone produced nonselec- i transduction pathways, such as signals that promote car- tive cation currents [7] as did polycystin-2 when expressed diac hypertrophy. In this respect, it is interesting to note in Sf9 cells [6]. These studies, together with our results, that patients suffering from ADPKD develop diastolic suggest that PKD2 and PKD2L2 are expressed in left dysfunction early in life, independent of additional risk ventricular myocytes and form functional ion channels in factors like hypertension [38,39]. 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 5 . Conclusion in single myocytes may actually result from a misdirected protein transport, either during the process of protein The present study shows that the LCC observed in rat synthesis or as a consequence of the isolation procedure. left ventricular myocytes display biophysical properties, 21 The LCC described in the present study displays bio- pharmacological profile, and regulation by [Ca ]i which physical and pharmacological properties very similar to are very similar to channels formed by polycystin-2, T. Volk et al. / Cardiovascular Research 58 (2003) 76–88 87 polycystin-L, and polycystin-2L2. Furthermore, single-cell [15] Callewaert G, Cleemann L, Morad M. Caffeine-induced Ca21 21121 RT-PCR analysis revealed that mRNAs of polycystin-2 release activates Ca extrusion via Na –Ca exchanger in cardiac myocytes. Am J Physiol 1989;257:C147–C152. and polycystin-2L2, but not polycystin-1 or polycystin-L [16] Rousseau E, Meissner G. 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