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

Transcription

1 Cardovascular Research 58 (2003) locate/ cardores A polycystn-2-lke large conductance caton channel n rat left ventrcular myocytes * Tlmann Volk, Alexander Peter Schwoerer, Susanne Thessen, Jobst-Hendrk Schultz, Hemo Ehmke* Insttut f ur Physologe, Unverstatskrankenhaus Hamburg-Eppendorf, Unverstat Hamburg, Martnstraße 52, Hamburg, Germany Receved 2 July 2002; accepted 17 December 2002 Abstract Objectve: Several members of the PKD gene famly (PKD2, PKDL and PKD2L2 ) are expressed n the heart. Polycystn-2 and ts homologues, whch are encoded by these genes, have recently been shown to form Ca -regulated nonselectve caton channels n heterologous expresson systems. Prevously, large conductance nonselectve caton channels (LCC) have been descrbed n cardomyocytes, however, ther molecular dentty remaned obscure. We therefore examned whether LCCs may be formed by polycystns. Methods: Myocytes solated from the rat left ventrcle were nvestgated by the whole-cell patch-clamp technque and sngle-cell RT-PCR. Results: Applcaton of 10 mm caffene to the bath soluton to ncrease the ntracellular Ca concentraton led to actvaton of LCC n 56% of the myocytes nvestgated (total n5651), n 10%, more than three LCCs were detected. The sngle channel conductance was 300 ps for monovalent catons and the channel was relatvely nonselectve for the monovalent catons Na, K, L, and Cs and 1 2 also permeable for the dvalent catons Ca and Ba, but mpermeable for NMDG and Cl. Amlorde (IC mm) and mllmolar concentratons of the trvalent catons Gd and La nhbted the LCC. Sngle-cell RT-PCR analyss revealed that mrna of PKD2 and PKD2L2, but not PKDL or PKD1 are expressed n ndvdual rat left ventrcular myocytes. Concluson: The characterstcs of LCC shown n the present study are nearly dentcal to those observed for polycystn-2 and ts homologues suggestng that polycystn-2 or polycystn-2l2 underle LCC n ventrcular myocytes European Socety of Cardology. Publshed by Elsever Scence B.V. All rghts reserved. Keywords: Ca-channel; Calcum (cellular); Ion channels; Myocytes; Na channel; Sngle channel currents 1. Introducton wth several subconductance states and were senstve to octanol. Based on these characterstcs, t was suggested Large conductance nonselectve caton channels (LCC) that the channels may be related to gap-juncton hemchanhave been descrbed n a number of preparatons ncludng nels [3]. LCC n rabbt ventrcular myocytes were found to the heart [1 4]. They have n common a relatvely large be permeable for monovalent as well as dvalent catons, sngle channel conductance ( ps), are permeable the sngle channel conductance was 380 ps (for monofor monovalent and dvalent catons but mpermeable for valent catons) and was reduced by ruthenum red or anons. In the heart, LCC have been descrbed and ryanodne. It was therefore suggested that the channel may characterzed n atral myoballs solated from the gunea be related to the ryanodne receptor (RyR) channel [4]. pg [3] and n ventrcular myocytes solated from rabbt However, several mportant questons remaned unanheart [4]. In atral myoballs, LCC dsplayed a sngle swered by these studes: gap-juncton hemchannels are channel conductance of 280 ps (for monovalent catons) usually closed under condtons under whch LCC have been recorded and t s unclear why and how the RyR channel should be present n the sarcolemmal membrane of *Correspondng authors. Tel.: (T. Volk); (H. Ehmke); fax: cardac myocytes. E-mal addresses: volk@uke.un-hamburg.de (T. Volk), ehmke@uke.un-hamburg.de (H. Ehmke). Tme for prmary revew 22 days /03/$ see front matter 2003 European Socety of Cardology. Publshed by Elsever Scence B.V. All rghts reserved. do: / S (02)

2 T. Volk et al. / Cardovascular Research 58 (2003) Recent studes suggest that a potental molecular cand- tentals. Whole-cell currents were low-pass fltered at 1 date for LCC may be formed by polycystn-2 or ts khz and sampled at 5 khz. Sngle channel current traces homologues polycystn-l and polycystn-2l2. Polycystns were fltered off-lne wth Hz usng the Gaussan are protens encoded by the polycystc kdney dsease procedure of the Patch-program (Dr Bernd Letz). Channel genes PKD1, PKD2, PKDL and PKD2L2, of whch open probablty (NP o) was calculated by dvdng the mutatons n PKD1 or PKD2 underle autosomal domnant ntegral of the current trace above the current level at polycystc kdney dsease (ADPKD; for revew, see Ref. whch all channels are closed by the sngle channel current [5]). Polycystn-2, polycystn-l, and polycystn-2l2 share ampltude and by the tme of analyss usng the Patch- 1 structural smlartes wth Na and Ca channels, and t program. Data are gven as mean6s.e.m., statstcal has been shown recently that polycystn-2 and polycystn- sgnfcance was calculated by the approprate verson of L form on channels whch are permeable for monovalent Student s t-test. Dfferences wth P,0.05 were consdered and dvalent catons wth conductances n the range of sgnfcant. Experments were carred out at room tempera ps when nvestgated n heterologous expresson ture ( C) f not stated otherwse. The nvestgaton systems [6,7]. Moreover, t has been shown that PKD2, conforms wth the Gude for the Care and Use of Labora- PKDL, and PKD2L2 are expressed n the heart [8 10]. We tory Anmals publshed by the US Natonal Insttutes of therefore questoned whether channels formed by Health (NIH Publcaton No , revsed 1996) and was polycystn-2, polycystn-l, or polycystn-2l2 may underle approved by local authortes. the LCC n the heart Solutons and chemcals 2. Methods Cardoplegc soluton contaned (n mm) NaCl 15, KCl 9, MgCl2 4, NaH2PO4 0.33, CaCl , glucose 10, 2.1. Patch clamp technque manntol 238, ttrated to ph 7.40 wth NaOH. Gga-ohm seals were obtaned n modfed Tyrode s soluton (control Experments were carred out on sngle myocytes so- soluton, n mm): NaCl 142, MgCl 1, NaH PO 0.33, lated from the left ventrcular free wall of female (unless CaCl2 1, glucose 10, Hepes 10, ttrated to ph 7.30 wth stated otherwse) Sprague Dawley rats ( g). NaOH. In some experments, 4 mm 4-amnopyrdne and Brefly, after nducton of anesthesa by.p. njecton of 0.1 mm BaCl2 were ncluded to nhbt K currents. To Trapanal (thopental-sodum, 100 mg/ kg body mass), the evaluate the permeablty for monovalent catons, the bath heart was quckly excsed and placed n cold (4 8C) soluton was smlar to control soluton except that t cardoplegc soluton. The aorta was cannulated and re- contaned 140 mm NaCl and was ttrated to ph 7.30 usng trogradely perfused for 5 mn wth nomnally Ca -free Trs. From ths soluton, ether 70 or 140 mm NaCl were modfed Tyrode s soluton, followed by 15 mn of perfu- substtuted by an equal amount of ether KCl, LCl, CsCl, son wth the same soluton contanng collagenase (type or N-methyl-D-glucamn-Cl (NMDG-Cl). Permeablty for CLS II, 200 U/ ml, Bochrom KG, Berln, Germany) and dvalent catons was estmated usng the followng bath protease (type XIV, 0.7 U/ ml, Sgma, St Lous, MO, solutons (n mm): NaCl 97, sucrose 100, glucose 10, USA). Fnally, the heart was perfused for 5 mn wth Hepes 10, ttrated to ph 7.30 wth Trs. NaCl and sucrose modfed Tyrode s soluton contanng 100 mm Ca. were substtuted by ether 97 CaCl 2, 97 BaCl2 or 97 After the perfuson, tssue peces from the left ventrcular MgCl 2. The ppette soluton contaned (n mm) CsCl 140, free wall were carefully removed usng fne forceps, MgCl2 5, EGTA 0.1, Hepes 10, Na2ATP 2, ttrated to ph further dsaggregated by pannng n modfed Tyrode s 7.20 wth CsOH (140CsCl soluton). In some experments, soluton contanng 100 mm Ca at 378C, and then 20 mm CsCl was replaced by 20 mm tetraethylam- 1 fltered through a cotton mesh. Isolated myocytes were monum-cl to nhbt K currents (120CsCl soluton). stored n modfed Tyrode s soluton contanng 100 mm When ndcated, the ppette soluton contaned 10 mm Ca. EGTA nstead of 0.1 mm to reduce the ntracellular The ruptured-patch whole-cell confguraton was used as concentraton of free Ca. In some experments, a Cs3- descrbed [11,12]. Membrane currents were recorded usng ctrate-based ppette soluton was used (n mm): ctrc acd an EPC-9 (Heka-Elektronk, Lambrecht, Germany) am- 65, CsCl 10, MgCl2 5, NaCl 2, Hepes 10, Na2ATP 4, ph plfer controlled by a Power-Macntosh computer (Apple 7.20 wth CsOH. For experments usng the perforated- Computer Inc., CA, USA) usng the Pulse software (Heka- patch confguraton, the ppette soluton contaned K-gluta- Elektronk). Ppette resstance averaged MV (n5 mate 110, KCl 10, NaCl 10, MgCl2 1, CaCl2 1, Hepes 5, 709) wth CsCl n the ppette, and MV (n520) amphotercn B 250 mm, ttrated to ph 7.20 wth KOH. wth K-glutamate n the ppette and control-soluton n the bath. The seres resstance ( MV, n5716) was 2.3. Sngle-cell RT-PCR compensated by 85%. Accordngly, at the largest recorded currents of about 1 na, the voltage error was less than 1 cdna synthess and sngle-cell RT-PCR were carred mv. VPp and Vm were corrected for lqud juncton po- out as descrbed prevously [13]. Brefly, under vsualza- 1

3 78 T. Volk et al. / Cardovascular Research 58 (2003) ton a sngle myocyte was sucked nto a mcroppette and maxmum shortly after actvaton of I Na/Ca, then declned transferred nto a reacton cup n whch, after short and eventually reached a steady-state or decreased to zero. centrfugaton and a freeze thaw cycle, reverse transcrp- The nset dsplays the frequency of observaton of the ton usng gene-specfc prmers followed. Subsequently, number of ndvdual channels n sngle cells. On average, two consecutve PCRs wth hemnested prmer pars were actvaton of sngle channel transtons of ths LCC was carred out. Prmer pars were ntron-overspannng to observed n 56% of myocytes nvestgated (total n5651); dentfy a possble amplfcaton of genomc DNA. PCR n %.1 ndvdual channels were dentfed. Smlar products were dentfed by sequencng. Postve controls results were also obtaned n myocytes solated from male for prmer effcency were run usng plasmds at several Sprague Dawley rats. In 50% of the myocytes (total dlutons (down to 0.1 fg plasmd DNA). PCR prmer n530), one or more LCC were present upon applcaton of sequences for rat PKD1, PKD2, PKDL, and PKD2L2 caffene to the bath soluton (data not shown). were: PKD1 upper prmer: 59-GGAGCGCTGGCCGGAG- Caffene opens the RyR channel and thus quckly ACCCTGG-39; PKD1 lower prmer: 59-TGGAGAGGCA- releases Ca from the SR [16], whch ncreases [Ca ] GGAAAGGTGTG-39. PKD1 upper nested prmer: 59-CG- approxmately up to 1 mm n rat ventrcular myocytes [15]. AGTCTGCGCATCCCGGCTGA-39. PKD2 upper prmer: The close assocaton of I and the actvaton of LCC Na/Ca 59-GGGACCCGCTGCATCGCCACC-39; PKD2 lower together wth the consecutve decrease n NPo after cessa- prmer: 59-CTCATAGAAAATAAAGCTCCGGTTGTC- ton of I suggest that an ncrease n [Ca ] s AG-39. PKD2 upper nested prmer: 59-CCGAGAGGCTG- responsble for actvaton of the channel. To further test GTGCGAGGAC-39. PKDL upper prmer: 59-GGCAGGC- ths hypothess, we carred out experments, n whch TCACAAGCTACAG-39; PKDL lower prmer: 59-CTCT- ntracellular Ca was buffered usng 10 mm EGTA n the CCCATCAGTCGGTTCAC-39. PKDL upper nested ppette soluton (n529). Fg. 2A shows a representatve prmer: 59-TTCAGGATCCAGACAAGCCAG-39. PKD2L2 recordng: upon applcaton of caffene, no I can be Na/Ca upper prmer: 59-GTCGTCCACGCTATCCCGCTG-39; observed and only rare openngs of LCC are noted. On PKD2L2 lower prmer: 59-CAACACAGGAACCAGCTA- average, NP o (calculated over a perod of 30 s after TGACC-39. PKD2L2 upper nested prmer: 59-AGCTTCG- applcaton of caffene n those recordngs, n whch CCATCATGTTCTTC-39. RT prmer sequences were: channel actvty was noted) was 30 tmes lower n the PKD2: 59-CGGCACTCCTAGCAGCAG-39; PKDL: 59- presence of 10 mm EGTA n the ppette soluton than n ACGTGTCTGGCTGCTGTAGG-39; PKD2L2: 59-GTTG- ts absence ( , n513 vs n539, TGTGAAATTTGTGAGCG-39. P,0.001), suggestng that Ca contrbutes to channel actvaton. Furthermore, an ncrease n extracellular [Ca ] from 1 to 10 mm resulted n an ncrease n NPo of LCC by a factor of 1765 (n519, P,0.05). 3. Results The presence of sngle channel actvty upon applcaton of caffene n the absence of an ncrease n [Ca ] could 3.1. Large conductance caton channels n left suggest that caffene may be requred to actvate the LCC. ventrcular myocytes Ths s further supported by the observaton that a sustaned actvty of LCC was noted n the presence of Ion channels formed by polycystn-2 and polycystn-l caffene n the bath soluton whch always ceased after have been shown to be actvated by an ncrease n the removal of caffene. To test whether LCC can also be ntracellular Ca concentraton ([Ca ] ). In the present actvated by an ncrease n [Ca ] n the absence of study, we used caffene to transently ncrease [Ca ]. caffene, a dfferent voltage-pulse protocol was used. Fg. 1 shows the effects of caffene on membrane currents Myocytes were stepped for 250 ms to VPp50 mvto recorded from three dfferent myocytes (A, B, C) at a actvate depolarsaton-nduced Ca -nflux/ SR-release contnuous holdng potental of VPp5290 mv. In Fg. 1A, and then back to VPp5290 mv to dentfy LCC. The the transent actvaton of an nward current s recorded ppette contaned 65 mm Cs3-ctrate, whch s known to after a delay of 5 s followng the swtch to caffene- decelerate the nactvaton of the L-type Ca current and contanng bath soluton. Ths transent nward current was to nduce regeneratve Ca -release from the SR thus not recorded n the presence of 5 mm N n the bath leadng to larger ntracellular Ca transents [17]. Fg. 2B soluton (data not shown) or when nternal Ca was dsplays a representatve recordng. Upon returnng V Pp buffered wth 10 mm EGTA (Fg. 2A), and has prevously back to 290 mv after the depolarsaton, actvaton of a 1 been dentfed as the Na / Ca exchanger current LCC can clearly be dentfed. Usng ths approach, LCC (I Na/Ca) [14,15]. Fg. 1B shows a smlar recordng n were detected n 30% of all cells nvestgated (n510). whch on top of INa/Ca a sngle channel wth a current These results ndcate that actvaton of LCC do not requre ampltude of 30 pa s actvated, whereas n another the presence of caffene. To address the possblty that LCC are artfcally formed durng the generaton of the whole-cell confgura- ton as a consequence of the rupture of the plasma recordng (Fg. 1C), actvaton of at least three dstnct sngle channels wth dentcal current ampltude can be dstngushed. Channel open probablty (NP ) reached a o Na/Ca

4 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 1. (A C) Effect of caffene (10 mm) on whole cell currents recorded from three dfferent myocytes. The ppette soluton contaned 120CsCl soluton wth 0.1 mm EGTA. The bath soluton contaned control soluton wth 4 mm 4-AP and 0.1 mm BaCl 2. In (A) a transent nward current s observed whch s carred by forward-mode I Na/Ca (see text). In (B) and (C), sngle channel transtons wth an ampltude of 30 pa were observed n addton to I Na/Ca. The nset ndcates the frequency of occurrence of the number of ndvdual channels (from a total of 651 recordngs). membrane, we carred out smlar experments usng the 3.2. Bophyscal propertes of the LCC perforated patch technque. Applcaton of caffene (10 mm) resulted n actvaton of the LCC n seven of eght In recordngs n whch LCC remaned actve for a attempts (data not shown) ndcatng that mechancal prolonged perod after cessaton of I Na/Ca, channel proprupture of the plasma membrane s not responsble for ertes were further nvestgated. Fg. 3A shows current LCC formaton or actvaton. traces wth sngle channel transtons recorded from one

5 80 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 2. (A) Smlar recordngs as n Fg. 1, except that the ppette soluton contaned 10 mm nstead of 0.1 mm EGTA. The horzontal bar ndcates the presence of caffene n the bath soluton. (B) Effect of depolarzaton-nduced Ca -nflux on membrane currents at VPp5290 mv n the absence of caffene. VPp was clamped for 250 ms to 0 mv and then back to VPp5290 mv. The ppette soluton contaned 65 mm Cs3-ctrate (see Methods). The bath soluton contaned control soluton. myocyte at holdng potentals between VPp5280 and 0 suggestng that the channel s ether less permeable for mv. Sngle channel ampltude progressvely decreased wth Cs than for Na, or s selectve for Cl wth an nwardly ncreasng V Pp. Myocytes could only be clamped to rectfyng characterstc. postve membrane potentals for a short perod (,1 s), Although the most often observed transton level was snce they hypercontracted upon the depolarzaton re- 300 ps, smaller conductance levels wth 150 or 75 ps sultng n a loss of the recordng. However, n experments were also occasonally noted. Fg. 3C shows a current n whch nternal Ca was buffered usng 10 mm EGTA, recordng n whch the smultaneous occurrence of two myocytes could be clamped to postve membrane po- conductance levels ( 300 and 150 ps) was noted. In fve tentals and outward sngle channel transtons were re- experments, the sngle channel ampltude was estmated at corded from VPp5130 to 190 mv. The average current 37 8C and averaged pa (VPp5290 mv). The voltage (I V ) relaton s shown n Fg. 3B. The outward resultng Q was 1.4, a value that has been found for 10 conductance was smaller than the nward conductance, many nonselectve caton channels (NSC) [18].

6 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 3. (A) Sngle channel transtons recorded at holdng potentals rangng from VPp5280 to 0 mv n a sngle left ventrcular myocyte. (B) Average I V relaton derved from 31 experments. Outward transtons were recorded n experments, n whch the ppette soluton contaned 10 mm EGTA. (C) Current trace demonstratng the smultaneous presence of two conductance levels. The sold lne ndcates the current level at whch all channels are closed, the dotted lnes ndcate the open states of the larger conductance level at a current ampltude of 30 pa. The ppette soluton contaned 140CsCl soluton wth 0.1 mm EGTA, the bath contaned control soluton and 10 mm caffene. 1 1 Fg. 4A shows current traces recorded at VPp5290 mv but slghtly less for Cs and L. Taken together, the LCC n the presence of 140, 70 or 0 mm NaCl n the bath. s selectve for catons over chlorde, but does only weakly Sngle channel ampltude decreased wth 70 mm NaCl n dscrmnate between monovalent catons. 1 the bath and when extracellular Na was completely The permeablty of LCC for dvalent catons was 1 replaced by NMDG, whch was only possble for bref nvestgated by ncreasng extracellular Ba, Ca or perods snce the myocytes contracted due to Ca entry Mg to 97 mm. All monovalent catons were removed 1 va the reverse mode of the Na / Ca exchanger, sngle from the bath soluton and osmolalty was mantaned by channel transtons became undetectable. Ths demon- addton of sucrose (100 mm). Wth 97 mm Ba n the 1 2 strates that the LCC s permeable for Na, but not for Cl. bath soluton, sngle channel ampltude averaged The correspondng average I V relatons are shown n Fg. pa (n58) at VPp5290 mv, whereas wth 97 mm Na, t 1 4B. Wth 140 mm Na n the bath (flled symbols), the averaged pa (n58). To avod contracton of average I V from 24 smlar recordngs revealed an myocytes, ntracellular Ca was buffered usng 10 mm average sngle channel conductance of 307 ps. Exchange EGTA n the ppette soluton when the bath soluton was of 70 mm of extracellular NaCl by the large organc caton swtched to 97 mm Ca. Under these condtons, sngle NMDG-Cl reduced the nward sngle channel ampltude to channel ampltude averaged pa (n53) at VPp5 about 50%. Both I V values were well ftted usng the 290 mv. In contrast, when the bath soluton was swtched 1 Goldmann-Hodgkn-Katz equaton wth the assumpton from 97 mm Na to 97 mm Mg, transtons dsappeared that the channel s selectve for monovalent catons. To (n56). These results demonstrate that the LCC s permedetermne the permeablty of LCC for other monovalent able for Ba and Ca, but not for Mg. catons, sngle channel ampltude was recorded at VPp52 1 To nvestgate the effects of dvalent catons on the 90 mv wth 70 mm extracellular Na replaced by an permeablty of the LCC for monovalent catons, extracel equmolar amount of ether NMDG, K, Cs or L lular Mg, Ca, or Ba was ncreased to 10 mm. Fg (Fg. 4C). The LCC s equally permeable for Na and K, depcts the effect of 10 mm Mg on the sngle channel 1

7 82 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 4. (A) Sngle channel current traces recorded at VPp5290 mv wth 140 mm Na (upper trace), 70 mm Na (mddle trace), or 0 mm Na (lower trace) n the bath soluton. (B) Correspondng average I V relatons recorded n the presence of 140 mm NaCl n the bath (flled symbols, n524) or after replacement of 70 mm NaCl by an equmolar amount of NMDG-Cl (open symbols, n54). I V relatons were ftted usng the Goldmann-Hodgkn-Katz equaton wth the assumpton of a selectve monovalent permeablty (see Results). (C) Sngle channel current ampltudes recorded at VPp5290 mv n the presence of 140 mm NaCl n the bath or after replacement of 70 mm NaCl by an equal amount of ether NMDG-Cl, KCl, CsCl, or LCl. ***P,0.001 versus 140 mm NaCl, n.s., not sgnfcant. The ppette contaned 140CsCl soluton wth 0.1 mm EGTA n all experments. conductance recorded at VPp5290 mv. In ths experment, mm amlorde on the LCC n a sngle recordng. In ths the ntal sngle channel ampltude was 29 pa, but when experment, amlorde reversbly reduced NPo by 72%. The the Mg concentraton was ncreased from 1 to 10 mm, nset dsplays a dose response relaton of the effect of sngle channel ampltude decreased by almost 50% to 17 amlorde on LCC, ftted by the Hll equaton. Half-maxpA. On average, sngle channel ampltude decreased from mal nhbton was observed at mm, whch s a control value of to pa (n58; P, smlar to that observed for polycystn-2 (27 79 mm) [6] ). Smlar results were obtaned for 10 mm Ba Gd and La, whch nhbt stretch actvated NSCs n ( vs pa; n55; P,0.01) and 10 mm mcromolar concentratons [20], and polycystn channels Ca ( vs pa; n512; P,0.0001). A and other NSC n the mllmolar range [6,7,19,], had no modulaton of monovalent caton permeablty by dvalent effect on NPo or sngle channel conductance of the LCC at catons has been reported prevously for polycystn-2 and 0.1 mm (n54 for Gd, n510 for La ), but rreversbly polycystn-l channels [7,19]. reduced NPo to zero at a concentraton of 1 mm (n56 for Gd, n58 for La ). Fg. 6B shows the effect of 5 mm 3.3. Pharmacology of the LCC N on LCC actvty. N reversbly reduced NPo by an average of 7369% (n512). To further characterze the LCC, the effects of several It has been suggested that a LCC observed n gunea pg substances known to affect polycystn channels and other atral myoballs may be related to gap juncton hemchan- NSC were nvestgated. Fg. 6A shows the effect of 500 nels [3]. We therefore tested the effect of the gap juncton

8 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 5. Sngle channel transtons of LCC recorded at VPp5290 mv n the presence of 1 mm (A) and 10 mm (B) extracellular Mg. The ppette contaned 140CsCl soluton wth 0.1 mm EGTA. The bath contaned control soluton. Ampltude hstograms were calculated over the whole current recordng depcted above. The sngle channel ampltude was estmated from the ampltude hstograms as well as by fttng equdstant current levels to the current recordngs. Both technques yelded the same current ampltude, ndcated below the current traces. hemchannel nhbtor 18-a-glycyrrhetnc acd (20 mm) Ths confrms the ntracellular presence of ryanodne and [22]. 18-a-glycyrrhetnc acd had no effect on ether NPo ts acton on the RyR channels. Taken together, these or sngle channel conductance (Fg. 6C, n511). results argue aganst the hypothess that the LCC n rat Ruthenum red, an nhbtor of RyR channels (and also of ventrcular myocytes s related to gap juncton hemchanother caton permeable channels [23]), reduced the sngle nels or the RyR. channel ampltude of a LCC n rabbt ventrcular myocytes [4]. We therefore nvestgated the effects of ruthenum red 3.4. Expresson of PKD1, PKD2, PKDL, and PKD2L2 (10 mm) n the bath soluton. In 15 of 16 experments, n ventrcular myocytes ruthenum red had no effect on sngle channel conductance. In one experment, we observed a shft to a lower Expresson of mrna of PKD1, PKD2 and ts homoconductance state. Ryanodne has also been reported to logues PDKL and PKD2L2 has been demonstrated n reduce the sngle channel ampltude of a LCC n rabbt murne and human whole heart preparatons [9,10,24]. To ventrcular myocytes when appled ntra- or extracellularly confrm the presence of all four mrnas n the rat heart, [4]. In our hands, ryanodne (10 mm) n the bath soluton RT-PCR wth prmers specfc for each gene was carred had no effect on NPo or sngle channel conductance (n5 out on total RNA solated from the rat left ventrcle. Fg. 13). Pre-ncubaton of myocytes wth ryanodne for up to 3 7A shows an agarose gel wth PCR products of the mn (n58) dd not prevent the LCC from actvaton nor expected length for PKD2, PDKL, and PKD2L2, and Fg. altered ts NPo or sngle channel conductance upon appl- 7B an agarose gel wth a PCR product of the expected caton of caffene. Also, n the presence of ryanodne (25 length for PKD1. All PCR products were dentfed by mm) n the ppette soluton, the sngle channel ampltude sequencng. These experments show that PKD1, PKD2 was smlar to that observed n the absence of ryanodne and ts homologues are expressed n the rat left ventrcle. (Fg. 6D, n532). On no occason dd we observe a shft to They do not exclude, however, the possblty that PKD1, a lower conductance level durng the course of the PKD2, PDKL, and PKD2L2 may only be expressed n experments. Applcaton of caffene after prolonged ncu- non-myocyte tssue such as blood vessels, connectve baton of myocytes wth ryanodne resulted n a reduced tssue, or cardac nerves. To test the expresson of PKD1, ampltude or complete absence of contracton and I Na/Ca. PKD2, PKDL, and PKD2L2 mrna n cardac myocytes

9 84 T. Volk et al. / Cardovascular Research 58 (2003) Fg. 6. (A) Effect of amlorde (500 mm) on LCC. On average, NPo decreased by 9763% (n59). The nset dsplays a concentraton response relaton of amlorde, half-maxmal nhbton was observed at mm. (B) Effect of N (5 mm) on LCC. (C) Effect of 18-a-glycyrrhetnc acd (20 mm) on LCC. (D) LCC actvty recorded after applcaton of caffene (10 mm) n the presence of 25 mm ryanodne n the ppette soluton. In ths recordng, caffene was appled 4 mn after breakthrough nto the whole-cell confguraton to allow for a suffcent dffuson tme for the ryanodne. The sngle channel ampltude was calculated usng the ampltude hstogram shown on the rght and averaged 30 pa. drectly, sngle-cell RT-PCR was carred out on solated 7C) and PKD2L2 (Fg. 7D). In total, PKD2 was detected left ventrcular myocytes. Fg. 7C1D shows representatve n seven of 20 (35%) myocytes and PKD2L2 n sx of 20 results obtaned by sngle-cell RT-PCR for PKD2 (Fg. (30%) myocytes. In contrast, nether PKDL (n520) nor

10 T. Volk et al. / Cardovascular Research 58 (2003) Dscusson 4.1. Mechansm of actvaton The close assocaton of LCC actvaton wth I Na/Ca upon applcaton of caffene to the bath soluton suggests that an ncrease n [Ca ] at least partcpates n actvaton of LCC. Ths s supported by the observaton that channel NPo was much lower when caffene was appled n the presence of EGTA n the ppette soluton. Furthermore, usng a ctrate-based ppette soluton, depolarzaton n- duced Ca -nflux/ SR-release transently actvated LCC n the absence of caffene thus demonstratng that an ncrease n [Ca ] alone s suffcent to actvate LCC. Smlar results have been obtaned n gunea pg atral myoballs: cyclc ncreases n [Ca ] observed wth a ctrate-based ppette soluton actvated LCC n the absence of caffene [3]. On the other hand, we consstently observed LCC actvty, although wth very low open probablty, upon applcaton of caffene n the presence of EGTA n the ppette soluton, suggestng that caffene alone (or together wth very low levels of [Ca ] ) can actvate LCC. Smlar results have been observed for LCC n rabbt ventrcular myocytes [4]. It therefore appears lkely that applcaton of caffene to the bath soluton actvates LCC by both an ncrease n [Ca ] and by a drect effect of caffene on the channel Relaton of the LCC to other cardac nonselectve caton channels In excsed nsde-out patches from solated ventrcular myocytes, Ca -actvated NSC wth a sngle channel conductance of 20 ps have been descrbed [26]. Smlar Fg. 7. (A,B) Ethdum bromde-staned gels of RT-PCR products, NSC have been detected n a varety of tssues (for revew, amplfed from total cardac RNA wth prmers specfc for PKD2, PKDL, see Ref. [18]). A potental molecular bass of these and PKD2L2 (A) and for PKD1 (B). (C,D) Ethdum bromde-staned gel channels may be the large famly of transent receptor of sngle-cell RT-PCR products, amplfed from ndvdual left ventrcular myocytes (1 5) wth prmers specfc for PKD2 (C) and PKD2L2 (D). potental (TRP) channels snce ths channel famly com- C1, C2, controls for each round of RT-PCR; RT, control for reverse prses members whch are Ca permeable NSCs (e.g. transcrpton; TC, control for surroundng tssue; M, molecular weght TRP1, TRP3 and TRP6) [27]. TRP channels are found n marker; 200 and 500 refer to the correspondng number of base pars. many tssues ncludng the heart, but they dffer from the LCC descrbed n the present nvestgaton wth respect to ther sngle channel conductance (whch s consderably lower), and pharmacology [28], thus makng t unlkely PKD1 (n570) were detected n any myocytes nvestgated. that the LCC belongs to the TRP channel famly. Smlar results were obtaned when the dentcal RT-PCR The rat LCC characterzed n the present study shares protocol was carred out on pools of 5 10 myocytes that many propertes wth two LCC whch were prevously were consecutvely sucked nto a mcroppette (n55). The descrbed n the hearts of gunea pgs and rabbts [3,4]. absence of PKDL mrna from myocytes s consstent wth These channels show a smlar sngle channel conductance, a recent study whch detected polycystn-l n the ep- permeablty for monovalent and dvalent catons, and cardum and n ventrcular blood vessels, but not n cardac regulaton by [Ca ]. The LCC descrbed n rabbt venmyocytes of the mouse [25]. Taken together, these data trcular myocytes was nhbted by ruthenum red and show that left ventrcular myocytes express PKD2 and ryanodne, and t was suggested that ths LCC may be PKD2L2, whereas PKDL and PKD1 expresson appear to related to the RyR channel [4]. Despte several smlartes, be lmted to non-myocyte cardac tssue. the LCC nvestgated n the present study was not affected

11 86 T. Volk et al. / Cardovascular Research 58 (2003) ether by extra- or by ntracellularly appled ryanodne, channels formed by polycystn-2 and ts homologues. All even though we used dentcal concentratons of ryanodne of them are permeable for mono- and dvalent catons, but and ts ntracellular acton was apparent by the absence of mpermeable for anons. The sngle channel conductance SR Ca release upon caffene applcaton after prolonged of polycystn channels [6,7] s well wthn the range we ncubaton wth ryanodne. Thus LCC n rat ventrcular have observed for the LCC. Both, LCC and polycystn myocytes appears to be smlar, but not dentcal to LCC channels, are actvated by an ncrease n [Ca ] and descrbed n cardac myocytes of gunea pgs and rabbts. nhbted by the trvalent catons Gd and La as well as amlorde at equal concentratons [6,7,19]. Furthermore, 4.3. Relaton of the LCC to on channels formed by both LCC n the present study and polycystn-2 channels polycystns are nsenstve to ryanodne [34]. Addtonal experments wth more specfc nhbtors of polycystn channels (whch PKD1 [29], PKD2 [24], PKDL [9], and PKD2L2 [10] are currently unavalable), or overexpressng polycystn-2 are expressed n the mouse and human heart, but t was or polycystn-2l2 n cardac myocytes are requested to unclear whether ths expresson actually occurs n car- further nvestgate the role of polycystns n cardac domyocytes. Usng sngle-cell RT-PCR, we could demon- myocytes. strate that n the rat heart, PKD2 and PKD2L2 are expressed n left ventrcular myocytes, whle the expres Possble functon of polycystns and LCC n the son of PKD1 and PKDL appears to be lmted to nonheart myocyte tssue. Prevous studes have shown that the expresson of channel mrna correlates wth the expres- The physologcal role of polycystns n cardac son of the correspondng on channel protens n cardac myocytes s stll unclear. Patents wth ADPKD are more as well as non-cardac tssue [30,31]. It therefore seems lkely to suffer from cardac abnormaltes such as septal lkely that polycystn-2 and polycystn-2l2 are also exdeformatons or mtral valve prolapse [8], suggestng a pressed n rat left ventrcular myocytes. In human empotental role of polycystns n the development of the bryonc hearts, polycystn-2 was detected n myocytes and heart. Ths noton s supported by a recent fndng that n the endocardum [32]. In contrast, a recent study mce wth homozygous deletons of PKD2 de n utero and nvestgatng the expresson of polycystn-2 n the rat heart dsplay severe cardac abnormaltes, partcularly septal faled to detect a sgnfcant proten level, whch, accordng defects [36]. Moreover, expresson levels of polycystn-2 to the authors, mght have resulted from a low senstvty are hgher durng embryonc development of the heart and of the antbody or from the preparaton technque [33]. decrease to lower levels at later stages of development An mportant queston s whether polycystn-2 and [24]. related protens are actually located n the plasma cell It has been suggested that polycystn-2 partcpates n membrane, or rather n ntracellular membranes such as the sgnal transducton pathways by modulatng [Ca ], pos- endoplasmc retculum. Recent studes suggested that sbly as a release channel [35,37]. Independent of the polycystn-2 may be prmarly located n ntracellular channel locaton, Ca -nduced Ca release s a tghtly membranes [34,35]. These results are supported by the controlled mechansm n cardac myocytes, and t s not observaton that a nonselectve caton conductance could qute clear whch role an addtonal large NSC should play. only be observed when polycystn-2 was expressed to- However, gven the relatvely long perods durng whch gether wth polycystn-1 [19], or when the proten transopenngs of the LCC can be observed after an ncrease n port from ntracellular compartments to the plasma mem- [Ca ], t seems possble that LCC may nfluence baselne brane was enhanced [34]. In contrast, expresson of [Ca ] and thus partcpate n Ca -medated sgnal polycystn-l n Xenopus oocytes alone produced nonselectransducton pathways, such as sgnals that promote cartve caton currents [7] as dd polycystn-2 when expressed dac hypertrophy. In ths respect, t s nterestng to note n Sf9 cells [6]. These studes, together wth our results, that patents sufferng from ADPKD develop dastolc suggest that PKD2 and PKD2L2 are expressed n left dysfuncton early n lfe, ndependent of addtonal rsk ventrcular myocytes and form functonal on channels n factors lke hypertenson [38,39]. the plasma membrane. The relatvely low abundance of channels that we have observed n the plasma membrane, may be explaned by a lack of PKD1 expresson n cardac myocytes. Alternatvely, the low number of LCC observed 5. Concluson n sngle myocytes may actually result from a msdrected proten transport, ether durng the process of proten The present study shows that the LCC observed n rat synthess or as a consequence of the solaton procedure. left ventrcular myocytes dsplay bophyscal propertes, The LCC descrbed n the present study dsplays bo- pharmacologcal profle, and regulaton by [Ca ] whch physcal and pharmacologcal propertes very smlar to are very smlar to channels formed by polycystn-2,

12 T. Volk et al. / Cardovascular Research 58 (2003) polycystn-l, and polycystn-2l2. Furthermore, sngle-cell [15] Callewaert G, Cleemann L, Morad M. Caffene-nduced Ca 1 RT-PCR analyss revealed that mrnas of polycystn-2 release actvates Ca extruson va Na Ca exchanger n cardac myocytes. Am J Physol 1989;257:C147 C152. and polycystn-2l2, but not polycystn-1 or polycystn-l [16] Rousseau E, Messner G. Sngle cardac sarcoplasmc retculum are expressed n ndvdual myocytes of the rat left Ca -release channel: actvaton by caffene. Am J Physol ventrcle. We therefore suggest that polycystn-2 or ts 1989;256:H328 H333. homologues underle LCC n rat left ventrcular myocytes. [17] Callewaert G, Spdo KR, Carmelet E, Pott L, Lpp P. Intracellular ctrate nduces regeneratve calcum release from sarcoplasmc retculum n gunea-pg atral myocytes. Pflugers Arch 1995;429: Acknowledgements [18] Semen D. Nonselectve caton channels. 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