iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 Contents lists ville t SieneDiret iohemil nd iophysil Reserh Communitions journl homepge: www.elsevier.om/lote/yr oth N- nd C-loes of lmodulin re required for C 2+ -dependent regultions of C V 1.2 C 2+ hnnels Feng Guo,, Etsuko Minoe, Kzuto Yzw, Hdhimuly smr, Xio-yn i,, Dong-yun Hn,, Li-ying Ho, *, Mski Kmeym, * Deprtment of Phrmeutil Toxiology, Shool of Phrmeutil Sienes, Chin Medil University, 92 eier Rod, Shenyng 111, Chin Deprtment of Physiology, Grdute Shool of Medil & Dentl Sienes, Kgoshim University, 8-35-1 Skurgok, Kgoshim 89-8544, Jpn rtile info strt rtile history: Reeived 28 Otoer 29 ville online 16 Deemer 29 Keywords: C 2+ hnnel Clmodulin Chnnel regultion Filittion ntivtion We investigted the onentrtion- nd C 2+ -dependent effets of CM mutnts, CM 12 nd CM 34,in whih C 2+ -inding to its N- nd C-loes ws eliminted, respetively, on the C V 1.2 C 2+ hnnel y inside-out pth lmp in guine-pig rdiomyoytes. oth CM 12 nd CM 34 (.7 1 lm) pplied with 3 mm TP produed hnnel tivity fter rundown. Conentrtion response urves were ell-shped, similr to tht for wild-type CM. However, there ws no ovious leftwrd shift of the urves y inresing [C 2+ ], suggesting tht oth funtionl loes of CM were neessry for the C 2+ -dependent shift. However, hnnel tivity indued y the CM mutnts showed C 2+ -dependent derese, implying C 2+ sensor existing esides CM. These results suggest tht oth N- nd C-loes of CM re required for the C 2+ -dependent regultions of C V 1.2 C 2+ hnnels. Ó 29 Elsevier n. ll rights reserved. ntrodution * Corresponding uthors. Fx: +86 24 23255471 (L.-y. Ho), +81 99 275 5522 (M. Kmeym). E-mil ddresses: lyho@mil.mu.edu.n (L.-y. Ho), kme@m.kufm.kgoshimu..jp (M. Kmeym). Voltge-gted L-type C 2+ hnnel hs een suggested to e involved in mny essentil proesses y the C 2+ -dependent regultion, inluding musle ontrtion, gene expression nd hormone seretion [1,2]. C 2+ -dependent filittion (CDF) nd intivtion (CD) hve een widely indited in those proesses [3 6]. Clmodulin (CM) is n importnt regultor of ion hnnels, serving s C 2+ sensor for C V 1.2 hnnel. Severl ritil moleulr determinnts, suh s Q, preq within the C-terminus nd region in the N-terminus of C V 1.2, hve een reported to intert with CM [4 1]. However, the regultions of CDF nd CD y C 2+ nd CM hve not een fully lrified. CM is protein formed y two loes, eh omprising two C 2+ -inding sites. CM mutted t C 2+ -inding sites 1 nd 2 in the N-loe (CM 12 ) nd t sites 3 nd 4 in the C-loe (CM 34 ) hve een used to investigte C 2+ -dependent regultion of C V 1.2 hnnel [1 13]. n spite of these efforts, the mehnism underlying loe-speifi regultion of CM of the C V 1.2 hnnel still remins to e estlished. n our previous study, we found tht C 2+ -free CM (pocm) ould produe oth filittory nd inhiitory effets on hnnel tivity with ell-shped onentrtion response reltionship, nd inresing [C 2+ ] shifted the urve towrd left (lower onentrtions). This shift ws not oserved with C 2+ -insensitive CM mutnt (CM 1234 ) [14]. n this study, we further explored the CM-medited regultion of the C V 1.2 hnnel with the CM mutnts (CM 12 nd CM 34 ). t is demonstrted tht oth N- nd C- loes of CM re required for the C 2+ -dependent regultions on the C V 1.2 C 2+ hnnel. Mterils nd methods Moleulr iology. Humn CM ws expressed s glutthione-strnsferse (GST) fusion protein in Esherihi oli L21, purified using glutthione Sephrose 4 (GE Helthre), nd the GST region ws leved y PreSission Protese (GE Helthre). The reovered CM ws quntified y the rdford method (Piere) with ovine serum lumin s stndrd. Point muttions in the C 2+ -inding sites of CM were introdued y Quikhnge site-direted mutgenesis kit (QGEN). CM 12 nd CM 34 hd muttions of E31 + E67 nd S11F + E14, respetively. Preprtion of single myoytes. Single ventriulr myoytes from dult guine-pig herts were dispersed y ollgense nd protese s desried previously [15,16]. Myoytes were treted with.5 mg/ml 1 protese (Ngse NK-13, Wko Pure Chemils, Osk, Jpn) nd.2 mg/ml 1 DNse (Type V, Sigm ldrih, St. Louis, US) to improve the suess rte of gig-ohm sel. Solutions. The solutions for single-hnnel reording were s desried previously [15,16]. The pipette solution ontined (mm): Cl 2 5, tetrethylmmonium (TE) Cl 7, EGT.5, y K 8644 6-291X/$ - see front mtter Ó 29 Elsevier n. ll rights reserved. doi:1.116/j.r.29.11.171
F. Guo et l. / iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 1171.3, nd Hepes CsOH uffer 1 (ph 7.4). The si internl solution ontined (mm): K-sprtte 12, KCl 2, gluose 1, EGT 2, nd Hepes-KOH uffer 1 (ph 7.4). The [C 2+ ] ws lulted using modified progrm of Fito [17]. Pth-lmp experiment. Single-hnnel urrents were reorded in the ell-tthed nd inside-out modes using pth-lmp mplifier (EPC-7, List, Drmstdt, Germny). C 2+ -hnnel urrents were eliited y depolrizing pulses from 7 to mv with 2 ms durtion t.5 Hz nd digitized t 3.3 khz. The vlue, where N ws the numer of hnnels in the pth nd Po ws the time-verged open-stte proility during the period of 5 15 ms, ws lulted for eh pulse. Chnnel tivity in ontrol ws otined y verging vlues for 2 min (6 pulses) in the ell-tthed ondition, nd those in test onditions were CM (3.5µM) + TP CM (1µM) + TP 4. 1nM [C 2+ ] 3. 1nM [C 2+ ] 3. 2. 2. 1. 1..5 2 4 6 8 1 12 14 16 18 2 2 4 6 8 1 12 14 16 18 2 CM (1µM) CM (3.5µM) 4. 1nM [C 2+ ] + TP 35 3 3. 25 2 2. 15 1 1. 5 1.4µM 3.5µM 1µM 2 4 6 8 1 12 14 [CM] (µm) 2. 1..5 e Chnnel tivity (%) 2 4 6 8 1 12 14 16 18 2 12 1 8 6 4 2 CM 12 (1.4µM) + TP 1nM [C 2+ ] CM 12 CM 34 CM 12 (1.4µM) CM 34 (1.4µM) 4. 1nM [C 2+ 3. ] 1nM [C 2+ ] 3. 2. 2. 1. 1..5 2 4 6 8 1 12 14 16 18 2 2 4 6 8 1 12 14 16 18 2 * * TP( + ) TP( - ) d d Chnnel tivity (%) CM 34 (1.4µM) + TP 3.5 1nM [C 2+ ] 3. 2. 1..5 2 4 6 8 1 12 14 16 18 2 Fig. 1. Effets of CM, CM 12, nd CM 34 on C V 1.2 hnnel tivity. () Filittory nd inhiitory effets of wild-type CM on C V 1.2 hnnel tivity. ( ) Chnnel tivity for repetitive depolriztion ws lulted s nd plotted ginst time. fter 2-min reording of in the ell-tthed mode, the inside-out pth mode () ws initited s indited y n rrow nd lsted for 1-min. Then, 3.5 lm CM (), 1 lm CM (), 1 lm CM followed y 3.5 lm CM (), together with 3 mm TP t 1nM [C 2+ ] were pplied t the times indited y the oxes. (d) Normlized hnnel tivity (s represented y m reltive to ontrol vlue) indued y 1.4, 3.5, nd 1 lm CM. Numer of myoytes for eh ondition ws 5. () Effets of CM 12 nd CM 34 on C V 1.2 hnnel tivity. 1.4 lm CM 12 (,) or CM 34 (,d) ws pplied with (,) or without (,d) 3 mm TP t 1 nm [C 2+ ] s indited y the oxes. lmost no effet ws seen without TP. (e) Summry of the effets of CM 12 nd CM 34 with (+) or without ( ) TP. *P <.5 ompred etween TP (+) nd ( ). Numer of myoytes for eh ondition ws 5.
1172 F. Guo et l. / iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 otined y serhing the mximum of men vlues (m)of onseutive 3-min periods (9 pulses) tken with one min intervl, nd normlized y the ontrol hnnel tivity. Dt nlysis. Dt were presented s mens ± SEM. Student s t-test ws used to evlute sttistil signifine, nd P <.5 ws onsidered to e signifint. Curve fitting for the [CM]-hnnel tivity reltionship ws performed with the softwre Deltgrph 5.4 ording to model, where mutully independent two CM-inding sites, one for filittion nd the other for inhiition, ws ssumed in our previous study [14]. Chnnel tivity () s n overll effet of CM would e: nf ½CMŠ Kd f 1 ¼ mx 1 þ ½CMŠ nf 1 þ ½CMŠ ni Kd f Kd i where mx is the mximum effet of CM, Kd f nd n f represent [CM] produing hlf effet nd the pprent Hill s oeffiient, respetively, for filittion, nd Kd i nd n i represent [CM] produing hlf effet nd the pprent Hill s oeffiient, respetively, for inhiition. Results Filittory nd inhiitory effets of wild-type CM on C V 1.2 hnnel tivity We first exmined the effet of wild-type CM on C V 1.2 hnnel tivity in the inside-out pth mode. Sine our previous studies suggested tht TP ws required for hnnel tivity, we pplied CM with 3 mm TP [18 2]. s shown in Fig. 1, hnnel tivity deresed rpidly fter pth exision in the si internl solution (rundown). Then pplition of 3.5 lm CM t 1 nm [C 2+ ]-indued hnnel tivity to level greter thn tht in the ell-tthed mode (274 ± 25%). However, s shown in Fig. 1, 1 lm CM + TP-indued hnnel tivity to smller level of ontrol (29.9 ± 11.1%). Fig. 1 indited tht the redued effet of 1 lm CM ws not due to the rundown sine the following pplition of 3.5 lm CM indued signifint effet. Thus, the effet of CM on C V 1.2 hnnel tivity ws iphsi s summrized in Fig. 1d. These results onfirmed our previous finding tht the onentrtion response reltionship of the CM s effet 5. 4. 3. 2. 1. CM 12 (1µM) CM 12 (3.5µM) 1nM [C 2+ ] + TP 2 4 6 8 1 12 14 Chnnel tivity (%) 1 8 6 4 2 [C 2+ ] 1nM 25nM 2µM.3 1 1 3 [CM 12 ] (µm) 5. 4. 3. 2. CM 34 (1µM) CM 34 (3.5µM) 1nM [C 2+ ] + TP 1. 2 4 6 8 1 12 14-7 mv, 2ms Chnnel tivity (%) 2p 14 [C 2+ ] 12 1nM 25nM 1 2µM 8 6 4 J 2.3 1 1 3 [CM 34 ] (µm) -7 mv, 2ms 2p Fig. 2. Conentrtion-dependent effets of CM 12 nd CM 34 on C V 1.2 hnnel tivity. () () fter 2-min ontrol reording in the ell-tthed mode followed y 1-min inside-out pth formtion (), 1 nd then 3.5 lm CM 12 were then pplied with 3 mm TP t 1nM [C 2+ ] s indited y the oxes. () Exemplr tres reorded t the time indited in (), for the ontrol ell-tthed mode (), 1 lm () nd 3.5 lm CM 12 (). () Normlized hnnel tivity (m reltive to ontrol) indued y.7 1 lm CM 12 with TP t fixed [C 2+ ] of 1 nm (lue), 25 nm (red) nd 2 nm (green). Numer of myoytes for eh ondition ws 5. Continuous lines re fitted urves with the eqution in Methods. () () Effet of 1 nd 3.5 lm CM 34 with protool similr to (). () Exemplr tres reorded t the time indited in (), for ontrol (), 1 lm () nd 3.5 lm CM 34 (). () Normlized hnnel tivity indued y.7 1 lm CM 34 with TP t three [C 2+ ] s indited. Numer of myoytes for eh ondition ws 5. Continuous lines re fitted urves with the eqution. (For interprettion of olor mentioned in this figure legend the reder is referred to the we version of the rtile.)
F. Guo et l. / iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 1173 on the hnnel showed ell-shped filittory nd inhiitory phse [14]. Effets of CM 12 nd CM 34 We then exmined effets of the CM mutnts, CM 12 nd CM 34, on C V 1.2 hnnel tivity. oth CM 12 (1.4 lm) nd CM 34 (1.4 lm) together with 3 mm TP t 1 nm [C 2+ ] produed hnnel tivity s shown in Fig. 1 nd, respetively. These effets were olished in the sene of TP (Fig. 1 nd d). Fig. 1e summrized the effets of CM 12 nd CM 34 on hnnel tivity with nd without TP. t ws found tht the CM mutnts CM 12 nd CM 34 ould produe C V 1.2 hnnel tivity in the presene of TP t low [C 2+ ], s shown for the wild-type CM. Conentrtion-dependent effets of CM 12 nd CM 34 To investigte onentrtion-dependent effets of the CM mutnts on C V 1.2 hnnel tivity,.7 1 lm of the CM mutnts were exmined with 3 mm TP t 1 nm [C 2+ ]. Fig. 2 nd showed tht first pplition of 1 lm CM 12 produed reltive hnnel tivity of out 3%, nd the following 3.5 lm CM 12 produed muh higher tivity of out 8% of ontrol. Similr experiments were repeted with different onentrtions of CM 12 (.7 1 lm) t [C 2+ ] of 1, 25, nd 2 nm, nd the normlized m vlues were plotted in Fig. 2. The urves t three different [C 2+ ] were ell-shped. The effets of CM 34 were lso iphsi (Fig. 2 nd ). The normlized m vlues, plotted ginst [CM 34 ] t [C 2+ ] of 1, 25, nd 2 nm, were lso ellshped (Fig. 2). These dt suggested tht CM 12 nd CM 34 produed oth filittory nd inhiitory effets on C V 1.2 hnnel tivity similr to the wild-type CM. However, it ws noted tht, unlike the wild-type CM, the urves for oth CM 12 nd CM 34 showed no ovious shift when [C 2+ ] ws hnged from 1 to 25 nm. C 2+ -dependent effets of CM 12 nd CM 34 To further exmine C 2+ -dependene effets of CM mutnts on the hnnel, [C 2+ ] ws hnged during pplition of the CM mu- 3.5 3. 2. 1..5 CM 12 (1.4µM) + TP 25nM [C 2+ ] 1nM [C 2+ ] 2 4 6 8 1 12 14 Chnnel tivity (%) 1 8 6 4 2 [CM 12 ] 3.5µM 1.4µM.7µM 1 1 1 1, 1, [C 2+ ](nm) 5. 4. 3. 2. -7 mv, 2ms CM 34 (1.4µM) + TP 25nM [C 2+ ] 1nM [C 2+ ] 1. 2 4 6 8 1 12 14 Chnnel tivity (%) 16 12 8 4 2p [CM 34 ] 3.5µM 1.4µM.7µM 1 1 1 1, 1, [C 2+ ](nm) -7 mv, 2ms 2p Fig. 3. C 2+ -dependent effets of CM 12 nd CM 34 on C V 1.2 hnnel tivity. () () fter 2-min ontrol reording in the ell-tthed mode followed y 1-min inside-out pth formtion (), 1.4 lm CM 12 with 3 mm TP were first pplied t 25 nd then 1 nm [C 2+ ] s indited y the oxes. () Exemplr tres reorded t the time indited in (), for ontrol (), CM 12 t 25 nm () nd 1 nm [C 2+ ] (). Normlized hnnel tivity indued y CM 12 of.7 (d), 1.4 (N), nd 3.5 lm (j) with 3 mm TP were plotted ginst [C 2+ ]. Numer of myoytes for eh ondition ws 5. () () Effet of 1.4 lm CM 34 with 3 mm TP t 25 nd then 1 nm [C 2+ ] s indited y the oxes with protool similr to (). () Exemplr tres reorded t the time indited in (), for ontrol (), CM 34 t 25 nm () nd 1 nm [C 2+ ] (). Normlized hnnel tivity indued y CM 34 of.7 (d), 1.4 (N), nd 3.5 lm (j) with 3 mm TP were plotted ginst [C 2+ ]. Numer of myoytes for eh ondition ws 5.
1174 F. Guo et l. / iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 tnts. n Fig. 3 nd, CM 12 (1.4 lm) ws first pplied t 25 nm [C 2+ ] nd then t 1 nm, resulting in slight inrese in m, n opposite effet seen with the wild-type CM. Similr result ws otined lso with CM 34 (Fig. 3 nd ). The result tht hnnel tivity deresed with inresing [C 2+ ] ws onfirmed for three onentrtions of CM 12 nd CM 34 (Fig. 3 nd ). These results suggested tht, lthough the onentrtion response urves for CM 12 nd CM 34 did not show C 2+ -dependent shift, it seemed tht C 2+ -dependent mehnism for the hnnel regultion still existed. C 2+ -dependene of the prmeters for CM 12 nd CM 34 effets The mximum hnnel tivity ( mx ) nd onentrtions for the hlf-mximum filittory nd inhiitory effets (Kd f nd Kd i ) of CM 12 nd CM 34 (Fig. 2 nd ) were estimted y urve fitting (see Methods) nd were plotted ginst [C 2+ ]infig. 4 nd, together with those for the wild-type CM [14]. The mx vlues for the CM mutnts were smller thn tht for the wild-type with less pronouned C 2+ dependene (Fig. 4). On the other hnd, the Kd f nd Kd i did not show onsistent hnge y hnging [C 2+ ] (Fig. 4), suggesting tht C 2+ -dependent hnge seen with the wild-type CM required intt oth loes of CM. Disussion n this study, the most intriguing finding is tht exogenously pplied oth CM 12 nd CM 34 n produe ell-shped onentrtion response reltionship. There is no ovious C 2+ -dependent shift in the onentrtion response urve for oth the CM mutnts, in ontrst to drmti C 2+ -dependent shift in the urve for the wild-type CM. Conentrtion-dependent effets of the CM mutnts on C V 1.2 hnnel tivity n our previous study, CM retivtes rundown hnnels in onentrtion-dependent mnner [19]. Furthermore, it hs een mx (%) CM WT 35 CM 12 3 CM 34 25 2 15 1 5 1 1 1 1, 1, [C 2+ ] (nm) Kd f nd Kd i C CM-depend. filittion 12 1 8 6 4 2 1 CM 12 Kd f CM 34 Kd f 1 CM 12 Kd i CM 34 Kd i 1 1, [C 2+ ](nm) C 2+ /CM-depend. filittion 1, Low or Moderte [CM] Low or Moderte [CM] nd Low [C 2+ ] nd Moderte [C 2+ ] CM-depend. intivtion d C 2+ -depend. intivtion (seondry mehnism) High [CM] nd Low or Moderte [C 2+ ] Low or Moderte [CM] nd High [C 2+ ] Fig. 4. C 2+ -dependene of the prmeters for CM 12 nd CM 34 effets nd hypothetil model for CM nd C 2+ regultions of C V 1.2 hnnel. () mx for CM 12 (4) nd CM 34 (h) plotted ginst [C 2+ ]. mx for wild-type CM tken from our previous study [14] is shown y roken line. () Kd f nd Kd i for CM 12 (4, N) nd CM 34 (h, j) plotted ginst [C 2+ ]. Kd f nd Kd i for wild-type CM tken from our previous study [14] re shown y hin nd roken line, respetively. (C) hypothetil model for the hnnel regultion. () t low or moderte [CM] nd t low [C 2+ ], the tivtion site () is prtly ound with pocm, wheres the inhiitory site () is CM-free, resulting in CM-indued hnnel tivity. () t low or moderte [CM] nd t moderte [C 2+ ], most of site is ound with C 2+ /CM ut site is still free, resulting in C 2+ /CMindued filittion of the hnnel. () t high [CM] nd t low or moderte [C 2+ ], most of nd sites re oupied y pocm or C 2+ /CM, resulting in pocm- or C 2+ / CM-indued intivtion of the hnnel. (d) t high [C 2+ ], lthough CM 12 or CM 34 n intert with site ut not with site, C 2+ inds diretly to the extr C 2+ -inding site presumly in the hnnel (), resulting in hnnel intivtion (seondry mehnism).
F. Guo et l. / iohemil nd iophysil Reserh Communitions 391 (21) 117 1176 1175 found tht CM nd CM 1234 show ell-shped onentrtion response reltionships [14]. n the present study, CM 12 nd CM 34 lso exhiit ell-shped reltionships, supporting the view tht the iphsi effets of CM my e intrinsi to pocm. The mximum tivity of the hnnel is produed y onentrtion of the CM mutnts t round 3 lm. This vlue is similr to tht of po- CM ut not of C 2+ /CM, implying tht CM 12 nd CM 34 ehve silly s pocm in the hnnel regultion. The tions of CM 12 nd CM 34 require TP, whih is onsistent with our previous findings tht TP is neessry for sl tivity of the hnnel [18 2]. However, moleulr mehnism underlying the tion of TP still remins to e lrified. C 2+ -dependent effet of CM lthough lrge numer of studies hve reveled importnt spets of the CM s role in CDF nd CD of the C V 1.2 hnnel [3 5,9 12], ontroversy still remins in the role of eh loe of CM. t hs een showed tht C 2+ -inding to the C-loe triggers rpid CD proess wheres the N-loe initites more grdul nd distint CD mehnism in C V 1.2 [1]. However, other studies suggest tht CD of C V 1.2 hnnel is ttriuted minly to the C- loe [11,12]. Moreover, it is lso indited tht C 2+ /CM-dependent protein kinse (CMK), ut not diret tion of CM, medites CDF [21,22]. n ttempt to explore the loe-speifi funtion of CM in CDF nd CD of C V 1.2, we hve exmined the C 2+ -dependent effet of CM 12 nd CM 34. oth Kd f nd Kd i vlues for the iphsi tion of CM 12 nd CM 34 do not show n ovious C 2+ dependene, whih is shown with the wild-type CM ut not with CM 1234 [14]. This suggests tht oth funtionl loes of CM re neessry for mnifesting the C 2+ -dependent effet of CM. Our findings my support the ide tht oth loes of CM prtiipte in CDF nd CD in C V 1.2 hnnels. CM-independent C 2+ regultion The present study hve found tht CM 12 nd CM 34 n derese mx in C 2+ -dependent mnner similr to tht oserved with CM 1234 [14]. t is therefore hypothesized tht there my e n dditionl C 2+ sensor esides CM. The ffinity of this sensor for C 2+ my e lower thn tht of CM, sine the derese in mx ours t higher [C 2+ ] thn those in Kd f nd Kd i whih re thought to reflet C 2+ -inding to CM. lthough we hve no evidene to identify this extr C 2+ -inding site, one possile ndidte my e region hving the -hnd-like motif in the proximl C-terminus of C V 1.2 C 2+ hnnel. Previous studies hve highlighted n importne of this -hnd region for CD, ut the onlusions remin ontroversil [23,24]. We speulte tht CM in the intivtion site my t s dominnt nd high-ffinity C 2+ sensor, wheres the postulted extr C 2+ sensor my t s seondry nd low-ffinity sensor, onstruting prudent nd sensile CD mehnism. hypothetil model for C 2+ -dependent regultion of C V 1.2 hnnel To explin the iphsi tion of CM, we hve proposed simple funtionl model, in whih two CM-inding sites, one for tivtion nd the other for intivtion, re ssumed in the C V 1.2 hnnel [14]. The present study extends this model (Fig. 4C). To postulte two CM-inding sites is further supported y the finding tht CM 12 nd CM 34 lso show iphsi effets on the hnnel. inding of CM (usully pocm) to the tivtion site produes hnnel tivity (Fig. 4C), nd the hnnel tivity is filitted y C 2+ - inding to CM (Fig. 4C). This filittion seems to require intt oth loes of CM, sine CM 12 nd CM 34 do not show this filittion. On the other hnd, the intivtion site hs lower ffinity for pocm, so tht pocm n ind to this site only t [pocm] higher thn the physiologil level (Fig. 4C). lterntively, when [C 2+ ]is elevted, C 2+ /CM would ind to the intivtion site with n inresed ffinity nd therey intivte the hnnel. gin this effet requires intt oth loes of CM. esides the CM s tion, there my e C 2+ -inding site tht triggers seondry inhiitory mehnism (Fig. 4Cd). We speulte tht the site my e the -hnd region or region tht interts with the downstrem inhiitory mhinery of the hnnel. Conlusions The present study revels some novel fetures of the CM-medited regultion of C V 1.2 hnnel: First, The effets of CM mutnts CM 12 nd CM 34 on hnnel tivity re ell-shped, supporting the hypothesis tht CM hs oth filittory nd inhiitory effet on the hnnel; Seond, unlike wild-type CM, neither CM 12 nor CM 34 shows C 2+ -dependent shift in the onentrtion response urve. These results suggest tht oth N- nd C-loes of CM re required for the filittory nd inhiitory effet on C V 1.2 hnnel tivity. knowledgments We thnk Ms. E. wski for seretril work on the mnusript nd Prof. Ji-qun Ci (Chin Medil Univ.) for ontinuous enourgement nd support. This work ws supported y reserh grnts from the Jpn Soiety for the Promotion of Siene to L.-Y.H. nd to M.K., nd from the Ntionl Nturl Siene Foundtion of Chin (367761, 38797) to L.-Y.H. Referenes [1] N. Tohse, S. Seki, T. Koyshi, M. Tsutsuur, M. Ngshim, Y. Ymd, Development of exittion ontrtion oupling in rdiomyoytes, Jpn. J. Physiol. 54 (24) 1 6. [2] W.. Ctterll, Struture nd regultion of voltge-gted lium hnnels, nn. Rev. Cell Dev. iol. 16 (2) 521 555. [3] F. VnPetegem, F.C. Chtelin, D.L. Minor Jr., nsights into voltge-gted lium hnnel regultion from the struture of the C V 1.2 Q domin-c 2+ / lmodulin omplex, Nt. Strut. Mol. iol. 12 (25) 118 1115. [4] R.D. Zühlke, G.S. Pitt, R.W. Tsien, H. 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