Endogenous calcium channels in human embryonic kidney

British Journal of Pharmaology (1996) 118, 748-754 1996 Stokton Press All rights reserved 7-1188/96 $12. 9 Endogenous alium hannels in human emryoni kidney (HEK293) ells Stanislav Berjukow, Frank Doring, Monika Froshmayr, Manfred Graner, Hartmut Glossmann & 'Steffen Hering Institut fur Biohemishe Pharmakologie, Universitat Innsruk, A 62 Innsruk, Austria 1 We have identified endogenous alium hannel urrents in HEK293 ells. Whole ell endogenous urrents (ISr-HEK) were studied in single HEK293 ells with 1 mm strontium as the harge arrier y the path lamp tehnique. The kineti properties and pharmaologial features of ISr-HEK were haraterized and ompared with the properties of a heterologously expressed himeri L-type alium hannel onstrut. 2 Isr-HEK ativated on depolarization to voltages positive of -4 mv. It had transient urrent kinetis with a time to peak of 16 + 1.4 ms (n = 7) and an inativation times onstant of 52 + 5 ms (n = 7) at a test potential of mv. The voltage for half maximal ativation was - 19. + 1.5 mv (n = 7) and the voltage for half maximal steady-state inativation was -39.7 + 2.3 mv (n = 7). 3 Blok of ISrHEK y the dihydropyridine isradipine was not stereoseletive; 1 gm (+) and (- isradipine inhiited the urrent y 3+4% (n=3) and 29+2% (n=4) respetively. (+)-Isradipine and (-)-isradipine (1 gim) inhiited ISr-HEK y 89 + 4% (n =5) and 88 + 8% (n = 3) respetively. The 7-romo sustituted (±)-isradipine (V265, 1 gim) whih is almost inative on L-type alium hannels also inhiited ISr-HEK (83+9%, n=3) as was oserved for 1 kim (-)-nimodipine (78+6%, n=5). Interestingly, 1 gim (±)-Bay K 8644 (n =5) had no effet on the urrent. ISr-HEK was only slightly inhiited y the one snail toxins wo-ctx GVIA (1 gm, inhiition y 17 + 3%, n = 4) and Cw-CTx MVIIC (1 gm, inhiition y 2 + 3%, n = 4). The funnel we spider toxin w-aga IVA (2 nm) inhiited ISr-HEK y 19+2%, n=4). 4 In ells expressing ISr-HEK, maximum inward urrent densities of.24 +.3 pa/pf and.39 +.7 pa/ pf (at a test potential of - 1 mv) were estimated in two different athes of HEK293 ells. The urrent density inreased to.88 +.18 pa/pf or 1.11 +.2 pa/pf respetively, if the ells were ultured for 4 days in serum-free medium. 5 Co-expression of a himeri L-type alium hannel onstrut revealed that ISr-HEK and L-type alium hannel urrents ould e distinguished y their different voltage-dependenies and urrent kinetis. The urrent density after heterologous expression of the L-type oa, suunit himera was estimated to e aout ten times higher in serum ontaining medium (2.14 +.45 pa/pf) than that of ISr HEK under the same onditions. Keywords: Ca2 + hannels; HEK293 ells; expression system; lass E alium hannels; T-type alium hannels; 1,4- dihydropyridines Introdution The human emryoni kidney ell line HEK293 serves as an effiient test system for funtional and iohemial studies of ioni hannel proteins. This ell line an e used as a transient or stale heterologous expression system and reently eame widely used for expression of voltage-dependent alium hannels (Williams et al., 1994; de Leon et al., 1995; Bangalore et al., 1995; Kamp et al., 1995; Shreiek et al., 1995; Perez- Garia et al., 1995). Many of the mammalian ell lines that are urrently used for expression of voltage-dependent alium hannels, as well as Xenopus laevis ooytes, have een shown to express endogenous alium hannels (Adams & Beam 1989; Bourinet et al., 1992; Skryma et al., 1994; Laerda et al., 1994). However, it is still unertain whether HEK293 ells express endogenous alium hannels. Preise knowledge aout the urrent density, voltage-dependene, kineti properties and pharmaologial features of endogenous urrents is a neessary preondition for studies of alium hannel al suunits or suunit ompositions expressed in these ells. In the present study we demonstrated that the ell line HEK293 does express endogenous alium hannels. We desrie their asi properties and suggest, that endogenous 1 Author for orrespondene at: Institut fur Biohemishe Pharmakologie, A-62 Innsruk, Peter Mayr StraBe 1, Austria. alium hannels in HEK293 ells have some kineti and pharmaologial similarities with lass E or T-type alium hannels (Soong et al., 1993). The endogenous alium hannels in HEK293 ells annot, however, e attriuted to a partiular lass of alium hannels (Birnaumer et al., 1994) eause the urrents are inhiited y 1,4-dihydropyridine ompounds with a profile that suggest an unusual non-speifi interation in omparison to L-type alium hannels. We demonstrate that the density of endogenous alium hannel urrents in HEK293 ells is inreased if the ells are grown in serum-free ulture medium. By transfeting HEK293 ells with DNA of a himeri L- type alium hannel al suunit, we illustrate that the urrents of endogenous and heterologously expressed L-type alium hannels an e separated y their voltage-dependene as well as y pharmaologial and kineti properties. Methods Cell ulture and transfetion HEK293 ells were grown at 37C in Duleo's modified Eagles medium (GIBCO, Grand Island, NY, U.S.A.) ontaining 1% foetal ovine serum (FBS, Seak, Suen, Austria) at 8% CO2. For differentiation HEK293 ells were ultured in

serum free (FBS-free) Optimem Medium (GIBCO) at 4-5% onflueny for 4 days. Two different athes of HEK293 ells were used in the present study. Experiments with the first ath (No. F-1626) of HEK293 ells were performed etween passage numers P34 to P42. The seond ath of HEK293 ells (No. F-1 1987) was studied etween passage numers P32 to P39. HEK293 ells were transfeted with a himera etween rait heart axca (H) (Mikami et al., 1989) and arp skeletal musle Ots (s) (Graner et al., 1991) sh (amino aid numers in parentheses): s(l-6), H(145-2171). As desried previously sustitution of the amino terminus of the aic-a suunit y a sequene of the arp skeletal musle al suunit resulted in a signifiantly S. Berjukow et al Endogenous alium hannels in HEK293 ells 749 higher expression rate of the resulting himera without affeting urrent kinetis (Wang et al., 1995). Heterologous expression experiments were routinely done with o-transfetion of the fla alium hannel suunit (Ruth et al., 1989). Calium hannel suunit DNAs were inserted into the mammalian expression vetor pdna3 (Invitrogen) and therefore expressed under the ontrol of a CMV promoter. HEK293 transfetions were mediated y Lipofetamin (GIB- CO) following the manufaturer's instrutions. A standard transfetion (in 35 mm Petri dishes) was done with 1 ug of eah expression plasmid, 9 yil Lipofetamin at a ell density of aout 7% onflueny. Path lamp experiments were performed etween 36 to 48 h after transfetion. a _4 --6 T1~~~~~~~~~~~. La T' U 1 6-2 2-2 4 2 pa 2 5 pa 1 Ms 1 Ms 4 6 8 d Memrane potential -6-4 -2 2 4 8 FBS free CL sh. Figure 1 Strontium inward urrents and urrent voltage relationship of 1Sr-HEK and ISr-sH in HEK293 ells: (a, ) Families of Isr HEK (ultured with 1% FBS) and ISr-sH in HEK293 ells. (a) Inward urrents (ISr-HEK) of a HEK293 ell grown for 4 days in DMEM supplemented with 1% FBS at voltages from -6mV to 4mV, in 2mV voltage steps, holding potential -7mV. () Inward urrents (ISr-sH) of a HEK293 ell (ultured in DMEM supplemented with 1% FBS) whih was transfeted with DNA enoding for the al himera sh (see Methods) at voltages from -2mV to 6mV, in 2mV voltage steps, holding potential -5mV (for partial inativation of 1Sr-HEK, see Figure 2). Test potentials are indiated. () Peak-urrent-voltage relationships of alium hannel urrents in HEK293 ells for ISr-HEK (same experiment as shown in a), ISr-HEK (ell was ultured in FBS-free medium) and ISr-sH (same experiment as shown in ). Smooth lines represent the est-fit urves to the funtion ISr-HEK/sH = Gmax*(V- Vrev)/(l + exp[(v-vo.5, at)/kat] 1sr-HEK/sH, values of the peak ISr-sH or 1sr-HEK of typial ells at a given test potential V; Gmax, maximal ondutane value; Vrev, reversal potential; VO.5,at, voltage of half-maximal urrent ativation; kat, slope fator. The estimated values for katt and VO.5,aCC are: '5r-HEK (1% FB5) (A) (kat= 7.53mV, V*5, at=-24.5 mv, Vrev =62 mv, G =.84 ps); isr-hek (FBS-free) (El) (kat =-6.7mV, V.5, at =-16.9mV, Vrev=46.5mV, Gmra,= 3.37pS); ISr-sH (O)(k =-5.84mV, V.5 at = 8.2 mv, Vrev = 68 mv, Gmax = 4.73 ps). The estimated urrent densities were.28 pa/pf (A\),.99 pa/pf (El) and 1.5 pa/pf (). (d) Current-voltage relationship otained y a 3 ms ramp depolarization from - 7mV to 8mV in a HEK293 ell 36 h after transfetion with DNA enoding for himera sh. Note that the two peaks of the inward urrent orrespond to the peak values of the urves otained in (). This protool enales the oservation of the expression of ISrHEK and ISr-sH in the same ell.

75 Ioni urrent reordings and data aquisition Inward strontium-urrents were reorded in HEK293 ells at 22-25C y the whole ell onfiguration of the path-lamp tehnique (Hamill et al., 1981) with a path lamp amplifier (EPC 7, List eletroni, Germany). Path pipettes with resistane of 1 to 4 Mohm were made from orosiliate glass (Clark Eletromedial Instruments, UK GC15-7.5) and filled with pipjette solution ontaining (in mm): CsCl 6, CsOH 6, aspartate 6, MgCl2 2, HEPES 1, EGTA 1 titrated to ph 7.25 with CsOH. Is, were measured in external strontium solution ontaining (in mm): SrCl2 1, N-methyl-D-gluamine 19, HEPES 1, gluose 2, 4-aminopyridine 4, tetraethylammonium hloride 27, MgCl2 3, uffered to ph 7.3 with methanesulphoni aid. Data were digitized using a DIGIDATA 12 interfae (Axon Instruments, Foster City, U.S.A.) and stored on the omputer hard dis. Data were filtered at 1 khz (four-pole Bessel filter) and digitized at a sampling rate of 5 khz. Leak urrents were sutrated digitally (using average values of saled leakage urrents eliited y a 1 mv hyperpolarizing pulse) or eletronially y means of an analogue iruit. Exhange of the ath solution (i.e. extraellular drug appliation) was ahieved y means of a miroperfusion hamer (MPC 5, List Eletroni, Germany) enaling repeated fast solution exhange in single ells without ontamination of the entire ath (see Savhenko et al., 1995 for details). This tehnique was highly suitale for appliation of small amounts of the peptide toxins as 5 pl test solution were suffiient for a 2 fold exhange of the hamer volume. Before drug appliation ells were monitored for run-down during a period of 1 min. Only ells with a run down of less than 5% were used to study toxin or drug effets. Data are given as ranges or means + s.e.mean. Statistial signifiane was alulated aording to Student's unpaired t test for two populations. S. Berjukow et al Endogenous alium hannels in HEK293 ells Results a 35 3 25 E 2 *mj 15 k._ 1 LO) 5 n -4-2 2 4 Memrane potential 6 Materials Human emryoni kidney (HEK293) ells were otained from the Amerian Type Culture Colletion (Rokville, MD, U.S.A.). The 1,4-dihydropyridines (+)- and (-)-isradipine and VO 265 (isopropyl 4-(2,3,1 (7-romo) enzoxadiazol-4- yl)- 1,4-dihydro-2,6-dimethyl-5-methoxyaronylpyridine 3- ar-oxylate) were kindly provided y Sandoz AG (Basle, Switzerland) and (±)-Bay K 8644 and (-)-nimodipine were from Bayer AG (Wuppertal, Germany). o-agatoxin IVA (Funnel We Spider toxin, o-aga IVA) was otained from BIO TREND (Germany) and w-onotoxin GVIA (marine snail toxin, o-ctx GVIA) was from Peninsula Laoratories (U.S.A.). o-conotoxin MVIIC was from Saxon Biohemials (Germany). tpeak = 16 ms tpeak - 32 ms Figure 2 Comparison of ativation and inativation kinetis of Isr HEK and ASr-sH in HEK293 ells. (a) Inativation time onstants of Is, HEK in non transfeted and ISr-sH in transfeted HEK293 ells are plotted as a funtion of the memrane potential. 1sr-HEK were eliited y 4ms depolarizing voltage steps from -7OmV to the indiated memrane potentials (ISr-HEK, (A) ells ultured in 1% FBS and ISr HEK, ([J) reorded after ulturing the ells for 4 days in FBS-free medium). Alternatively ISr-sH () were reorded during 4ms depolarizations from -5 mv. The inativation time ourse was fitted y a single exponential funtion. Data from three representative ells. (,) Solid lines represent est fit to the funtion ISr-HEK/sH (t) = A*exp(- t/tinat) + C. For 1Sr-HEK () Tinat = 6 ms (at mv) and for 'Sr-sH () Tinat = 132 ms (at 2 mv) were estimated. Tale 1 Current type Endogenous alium hannel urrents were oserved in 16 (76%) out of 21 non-transfeted HEK293 ells with 1 mm strontium as the harge arrier. Depolarizing the ell memrane from a holding potential of -7 mv aove a threshold of -4 mv evoked transient voltage-dependent inward urrents. Figure la shows a family of whole ell ISr-HEK from a nontransfeted HEK293 ell. The urrents reahed maximal ISr-HEK- (1% FBS) ISr-HEK* (FBS-free) ISr-sH* (1% FBS) Properties of ISr-HEK and ISr-sH in HEK293 ells Current density (pa/pf) VO.5,at kat VO.5,inat kinat. G(,S (ps) tpeak (ms).24 +.3** -19. ± 1.5 5.7 +.35-39.7 ± 2.3-3.6 +.3.82.12 16.8 ±1.4.88±.18** -18.5± 1.7 5.6±.1-41.2± 1.5-3.7±.27 3.1 ±.2 17.± 1.2 2.14 ±.45** 9.2± 1.1 5.2±.8-1.1 ± 1.3-5.7±.36 4.85 ±.93 34.± 1.1 Tinat (ms) 52± 5 57 + 5 29 ± 2 Current kinetis of ISr-HEK and Isr-sH were studied at test potentials of mv and 2 mv respetively. For Isr-HEK measurements the holding potential was -7 mv. To minimize ontamination of ISrsH y Isr-HEK transfeted ells were held at -5 mv if urrent kinetis of ISr-sH were studied. Areviations: kat/ina, slope fator of orresponding steady-state ativation or inativation urves; VO.5,at/inat potentials of half-maximal urrent ativation and inativation; ;inat, inativation time onstant, tp1,, time to peak of the inward urrent. Values are the mean ± s.e.mean from 5 to 7 HEK293 ells. *Cells from ath No.F-1626, passage numers P34 to P42. **The oserved differenes in urrent densities were highly signifiant: ISr-HEK (1% FBS) versus ISrHEK (FBS free) P<.1.

values at -6 + 1. mv (n = 7) and reversed at 55 + 6.1 mv (n = 7). Voltages for half maximal ativation were estimated as desried in Figure I (see Tale 1). ISTHEK reorded y voltage ramp protools in nontransfeted HEK293 ells had a single peak with no evidene for more than one alium hannel type expressed and a similar urrent-voltage relationship to that oserved in Figure l (data not shown). The time ourse of urrent inativation during a 4 ms test depolarization was approximated y a single exponential funtion (Figure 2). The alulated inativation time onstant was voltage-dependent, delining in the ell shown in Figure 2 from 117 ms at -2 mv to 34 ms at 3 mv (Figure 2a). ISr- HEK ativated rapidly reahing a time to peak etween 14.8 ms and 18.8 ms at mv (Figure 2, see Tale 1 for mean values of time to peak at a test potential of mv). The voltage-dependene of alium hannel availaility was determined y measuring the peak ISrHEK during a test pulse to mv after a 1 s onditioning prepulse (see Figure 3). The voltage for half maximal inativation of ISr-HEK is indiated in Tale 1. Redution or omplete removal of growth fators from the ulture medium indues in many ell lines a morphologial differentiation and aumulation of speifi gene produts like alium hannel proteins (see Biel et al., 1991). We have grown HEK293 ells for 4 days in FBS-free ulture medium and oserved a signifiant inrease in Isr-HEK-density whereas the voltage-dependene and kinetis of alium hannel urrents remained unhanged (Figure i, see Tale 1 for data from ell ath No. F-1626). In a seond ath of HEK293 ells (see Methods) the urrent density inreased from.39 +.7 pa/pf (n = 26) to 1.11 +.2 pa/pf (n = 15) after removal of FBS from the ulture medium. In previous studies L-type al suunits have een expressed in the HEK293 ells ut no endogenous alium hannel urrents have een reported (i.e. Perez-Garia 1993; Shreiek et al., 1995). We have onsidered the possiility that endogenous alium hannels may appear only in ertain ell athes and therefore performed experiments on two different athes of HEK293 ells with different passage numers (see Methods). Voltage-dependene and urrent kinetis of ISr-HEK were found to e similar in oth HEK293 athes tested. To ompare voltage-dependene and kineti properties of ISr-HEK with L-type alium hannels we transfeted HEK293 ells with DNA enoding for the himeri al suunit sh (see also Wang et al., 1995; Graner et al., 1996; for kineti and S. B o a Berjukow et al Edeulmnl Endogenous alium hannels in "I HEK293 ells 751 5 pharmaologial properties of onstrut sh). Figure l shows ISr-sH of a HEK293 ell reorded 48 h after transfetion with the orresponding DNA. ISrH ativated and inativated with slower urrent kinetis ompared to ISr-HEK (Figure la). I'r-sH ativated during depolarizing steps from -5 mv aove a threshold of -2 mv and reahed maximal urrent values at 2 + 2 mv (n = 7). The urrent voltage relationship of ISr-sH is shown in Figure I (same ell as in Figure l, superimposed with the ISrHEK urrent voltage relationships of nontransfeted HEK293 ells). The fat that two hannel types (ISr-HEK and Isr-sH) with different voltage-dependene are expressed in a single HEK293 ells was also evidene from the urrent-voltage relationship. When ramp depolarizations were applied from a holding potential of -7 mv to + 8 mv (see Figure Id) two peaks were distinguishale. Similar oservations were made in 26 (71%) out of 37 ells expressing the L-type himera sh. The first peak on the urrent-voltage relationship (Figure ld, related to ativation of ISrHEK) ould e redued y holding the ells at more depolarized potentials (data not shown). At later stages after transfetion (>48 h) a separate peak of ISr-HEK on the urrent-voltage relationship was less prominent and presumaly superimposed y threshold ISr-sH eause this hannel type was expressed at a muh higher density (see Tale 1). The voltage-dependene of steady state inativation and kineti properties of ISr-sH and ISr-HEK are shown in Figure 3. Compared to 'Sr-HEK, the voltage for half maximal inativation of ISr-sH was found to e aout 3 mv more positive (Tale 1, see also Figure la,, for the different thresholds of ISr-sH and ISrHEK ativation). Inativation kinetis of I5r-sH were desried y a voltage-dependent time onstant deaying from 276 ms at -2 mv to aout 45 ms at 4 mv (Figure 2a, ; see Tale 1 for mean values, of time to peak and inativation time onstants at 2 mv test potential). The maximal urrent density of ISr-sH in transfeted ells was estimated from the peak urrent values otained upon depoa 1. L C14 L ~~~~~~~~~~~~~CN a) J C.) a).n V z.5 F 1 _1_ 1 ms d 1 ms KI Lfl. -1-8 -6-4 -2 Prepulse potential 2 4 5 ms Figure 3 Steady state inativation urves of isr-hek and Ir-sH: the peak urrents during a 3ms test pulse to OmV (ISr-HEK, ) or 2mV (ISr-sH, ) were plotted versus the voltage of a los onditioning prepulse. Interpulse interval was 2ms. Solid lines are drawn aording to the equation: ISr-HEK/ISr-HEK max = 1/1(1 + exp[(v- V.5, inat)/kinaj; ISr-HEK: Vo.5, inat =- 37.5mV; kinat = -3.67 mv; Isr-sH: VO.5inat= - 12.5 mv, kinat = -5.95 mv. Inset: 1sr-HEK, () reorded after onditioning prepulses to -85, -7, -55, -4, -25, -1 and 5mV, aliration ars 5OpA and looms. ISr-sH, () reorded after ondition prepulses to -6, -5, -4, -3, -2, -1,, 1 and 2 mv, aliration ars 5 pa, looms. Figure 4 Stereoseletivity of isradipine ation on ASr-HEK and ISr-sH* HEK293 ells were depolarized every lo s from a holding potential of -7mV to a test potential of mv (for oservation of IAr-HEK) and to 2mV (for oservation of ISr-sH). Currents in the asene (1) and presene of drug (2), indiating Isrinhiition in steady state, are superimposed. Experiments were performed on ells ultured in the presene of 1% FBS. (a) Inhiition of ISr-sH (1-ontrol) y 1 FM (+)-isradipine (2) and () lok of ISrsH (1-ontrol) y 1 IM (-)- isradipine (2). () Inhiition of ISHEK (1) y 1 ym (+)-isradipine (2) and (d) 1sr-HEK (1) inhiition y 1 Mm (-)-isradipine (2).

752 larization of the ell memrane from a holding potential of -5 mv to a test potential of 2 mv. The estimated values of the urrent density ranged from 1. up to 3.5 pa/pf (see also Tale 1 for mean values). ISr-sH were sensitive to 1,4-dihydropyridines and inreased more than 3 fold (mean of 3 experiments) at a test potential of 2 mv upon appliation of 1 gm )(±)-Bay K 8644, whereas appliation of (± )-Bay K 8644 (up to 1 um) did not affet ISr HEK- ISr-sH were stereoseletively inhiited y the antagonist isradipine (Figure 4a, ). (+)-Isradipine (1 gum) loked ISr-sH y 87 + 2% (n = 3) whereas the (-)-enantiomer was signifiantly less effetive (with P<.1) and indued only 61 + 3% (n = 3) lok. Pharmaologial features of ISr-HEK were studied at a holding potential of -7 mv during depolarizing steps to mv in HEK293 ells ultured in the presene of 1% FBS. Isradipine inhiited ISr-HEK ut the lok was not stereoseletive (Figure 4, d). (+)-Isradipine (1 jim) indued a 3 + 4% (n = 3) lok whereas (-)-isradipine inhiited ISr-HEK y 29 + 2% (n = 4). A higher onentration (1 jgm) of (+ )- and (-)-isradipine loked ISr-HEK y 89 + 4% (n = 5) and 88 + 8% (n = 3) respetively. (-)-Nimodipine (1 jgm) indued a similar amount of peak urrent inhiition (92 + 3% (n = 5)). The lok y isradipine was reversile (washout after 1 min 79+16% (n=6)), whereas lok y (-) nimodipine was nearly irreversile (after 1 min washout less than 2% reovery). The 7-romo sustituted (±)-isradipine (VO 265, 1 gim) that is almost inative on L-type alium hannels (Glossmann & Ferry, 1985) also inhiited ISr-HEK (lok y S. Berjukow et al Endogenous alium hannels in HEK293 ells 83+9%, n=3). Endogenous alium hannel urrents were inhiited y Ni2+ with half maximal lok ourring at 48+19 gim (n=4) (Figure 5a). The one snail toxin o-ctx MVIIC (1 gm) indued 2 + 3% (n = 4) lok Of ISr-HEK (Figure 5) whereas o-ctx GVIA (1 gum) indued 17+3% (n=4) urrent inhiition (Figure 5). The funnel we spider toxin o- Aga-IVA (2 nm) indued only weak inhiition (19 + 2%, n = 4) of the endogenous alium hannel urrent (Figure 5d). Disussion The main purpose of the present study was to haraterize the HEK293 ell line as an expression system for voltage-dependent alium hannels. We estalished that HEK293 ells, as previously shown for other expression systems, possess endogenous voltage-dependent alium hannel urrents. The perentage of HEK293 ells expressing ISr-HEK was even higher (76%) ompared to CHO ells where only 24% of the ells were found to express endogenous alium hannels (Skryma et al., 1994). The studied ISr-HEK have a transient time ourse and are insensitive to the L-type alium hannel agonist (± )- Bay K 8644 (1 jim). Similar pharmaologial features, inluding the weak inhiition of o-aga-iva (Soong et al., 1993, ut see Ellinor et al., 1993), o-ctx GVIA (Soong et al,. 1993, ut see Ellinor et al., 1993) and o-ctx MVIIC (Ellinor et al., 1993) have een previously desried for lass E alium hannels (Williams et al., 1994). In addition to its ommon pharmaologial properties, the fast inativation time ourse a IQ LO) CNI 4 3 2. o 2 25 ms 1 25 ms d CL CNI CNI 2 1 25 ms 25 ms Figure 5 Pharmaologial features of ISr-HEK (a) Inhiition of 1sr-HEK (1) y susequent appliation of 3Mm (2), 3Mm (3) and 1 Mm (4) nikel. () Inhiition of ISr-HEK (1) y 1 MM o-ctx MVIIC (2); () Inhiition of 1Sr-HEK (1) y 1 MM Cw-CTx GVIA (2); (d) inhiition of ISr-HEK (1) y 2 nm o-aga-iva (2). 1Sr-HEK were evoked y depolarizations from - 7mV to mv applied at a frequeny of.1 Hz.

S. Berjukow et al Endogenous alium hannels in HEK293 ells 753 Of ISr-HEK is also typial for lass E alium hannels (Soong et al., 1993; Ellinor et al., 1993). Class E alium hannels inativate with a time onstant of aout 7 ms (15 mm arium as harge arrier, Williams et al., 1994) ompared to ISr-HEK inativating with 1 mm strontium as harge arrier with a time onstant of aout 6 ms in the voltage range of -1 to 3 mv (see Figure 2a, ). The onentration for half maximal inhiition of ISr-HEK (see Tale 1) y Ni2+ (IC5=48 gim) also orresponds to the reported half maximal inhiition of lass E alium hannels (IC5 for urrent lok y Ni2+ 3 gm, Soong et al., 1993; Ellinor et al., 1993; Williams et al., 1994). Taken together, with respet to the estalished degree of identity in the pharmaology and kineti properties, endogenous alium hannels in HEK293 ells share some properties with lass E alium hannels (Soong et al., 1993; Birnaumer et al., 1994). In previous studies lass E alium hannels have een reported to e insensitive to the 1,4-dihydropyridines, nifedipine (1 pm, Soong et al., 1993) and nimodipine (1 gm, Ellinor et al., 1993). The inhiition y isradipine and (-)-nimodipine distinguishes ISr-HEK from lass E hannels. Inhiition of ISr HEK y isradipine was, however, not stereoseletive and ourred at higher onentrations ompared to the stereoseletive lok of ISr-sH y the same drug (Figure 4). Additionally the 7- romo-ester-sustituted (± )-isradipine (V265) also inhiited ISr-HEK. This 7-romo-ester-sustitution (as in V265) is detrimental for L-type alium hannel inding (Glossmann & Ferry, 1985; Janis et al., 1987). Thus, 1,4-dihydropyridine inhiition of ISr-HEK learly differs from that oserved for L- type alium hannels. Taken together, the endogenous alium hannel in HEK293 ells shares many harateristis with a low voltageativated lass E alium hannel or T-type hannel desried y Soong et al. (1993). However, this hannel type does not fit preisely into any lass of voltage-dependent alium hannels as defined y Birnaumer et al. (1994), eause of the following pharmaologial properties: (i) non-stereoseletive (ut reversile) inhiition y isradipine and inhiition y (-)-nimodipine; (ii) no effet of (± )-Bay K 8644 and (iii) inhiition of ISr HEK y (±)-V265 with similar poteny to that of isradipine. Clearly, this pharmaologial profile for dihydropyridine ompounds differs from that known for L-type alium hannels and further studies will show if endogenous alium hannels in HEK293 represent a new lass of voltagedependent alium hannels. The important finding of this study is that whole ell alium hannel urrents of expressed exogenous al suunits in HEK293 ells an e ontaminated y endogenous urrents. The degree of ontamination is dependent on the relation etween the urrent densities of ISr-HEK and the expressed L-type hannel. Shreiek et al. (1995) report L-type urrent densities in HEK293 ells (IBa measured with 3 mm arium as harge arrier) of more than 2 pa/pf if L-type al suunits are expressed alone, and up to 2 pa/pf when other alium hannel suunits are o-expressed (ompare with the urrent densities summarised in Tale 1). However, as most studies were performed under different experimental onditions with respet to the hosen harge arrier (divalent ation), its onentration, and the time interval after transfetion when urrents were studied, it appears to e diffiult to ompare the oserved urrent densities otained in different laoratories. Our data indiate, however, that it is possile to study funtional properties of L-type al alium hannel proteins in HEK293 ells: (i) First of all, the urrent density of L-type urrents reorded 48 h after transfetion ranged aout one order of magnitude aove the urrent density oserved in nontransfeted HEK293 ells whih were ultured in a medium supplemented with 1% FBS. It has, nevertheless, to e emphasised that the density of endogenous alium hannels in HEK293 ells is dependent on the ell ulture onditions and sustantially inreased if the ells are ultured in FBS-free ulture medium (see Tale 1). (ii) Beause 'Sr-HEK have a lower voltage for urrent ativation, a possile ontamination of expressed L-type alium hannels y 1Sr-HEK an e sustantially redued if the memrane potential is held in the range -4 to -35 mv (see Figure 3) or orresponding prepulses are applied. Under these onditions more than 5% of the ISrHEK are in an inativated non availale state whereas L-type, alium hannel urrents (ISr-sH) are ompletely availale (Tale 1, Figure 3). Furthermore, a ontamination with 'Sr-HEK an e oserved at early stages after transfetion if ramp depolarizations are applied (Figure ld) and ells with a high density of 1SrHEK an e exluded from analysis. (iii). Finally, the 'ontamination' of whole ell L-type alium hannel urrents y 'Sr-HEK is dependent on the test pulse potential and will e small in the range of 1 to 3 mv eause ISr-HEK reah peak urrent values at more negative voltages (see Figure 1). Consequently, a ontamination of L-type urrents y endogenous alium hannel urrents in HEK293 ells an e minimised if the experimental onditions (i.e. voltage protools, ell ulture onditions) are optimised and L-type alium hannels are expressed at high enough density. We thank Prof. A. Shwartz for providing the alc-a DNA, Dr D. Beeh and Dr E.N. Timin for helpful suggestions and B. Kurka for expert tehnial assistane. This work was supported y grants from the FWF S 661 (H.G.) and S 663 (S.H.). Referenes ADAMS, B.A. & BEAM, K.G. (1989). A novel alium urrent in dysgeni skeletal musle. J. Gen. Physiol., 94. 429-444. BANGALORE, R., MEHRKE, G., GINGRICH, K., HOFMANN, F. & KASS, R.S. (1995). Gating urrents of reominant ardia L-type alium hannels expressed in human emryoni kidney (HEK293) ells: relationships etween harge movement and ioni urrent. Biophys. J., 68, A14. BIEL, M., HULLIN, R., FREUNDNER, S., SINGER, D., DASCAL, N., FLOCKERZI, V. & HOFMANN, F. (1991). Tissue speifi expression of high-voltage-ativated dihydropyridin-sensitive L-type alium hannels. Eur. J. Biohem., 2, 81-88. BIRNBAUMER, L., CAMPBELL, K.P., CATTERALL, W.P., HARPOLD, M.M., HOFMANN, F., HORNE, W.A., MORI, Y., SCHWARTZ, A., SNUTCH, T.P., TANABE, T. & TSIEN, R.W. (1994). The naming of voltage gated alium hannels. Neuron, 13, 55-56. BOURINET, E., FOURNIER, F., NARGEOT, J. & CHARNET, P. (1992). Endogenous Xenopus-ooyte Ca-hannels are regulated y protein kinases A and C. FEBS Lett., 299, 5-9. DE LEON, M., JOHNS, L., PEREZ-REYES, L., WEI, X., SOONG, T.W., SNUTCH, T.A & YUE, D.T. (1995). An essential strutural domain for Ca-sensitive inativation of L-type Ca hannels. Biophys. J., 68, A13. ELLINOR, P.T., ZHANG, J-F., RANDALL, A.D., ZHOU, M., SCHWARZ, T.L. & TSIEN, R.W. (1993). Funtional expression of a rapidly inativating neuronal alium hannel. Nature, 363, 455-458. GLOSSMANN, H. & FERRY, D.R. (1985). Assay for alium hannels. Methods Enzymol., 19, 513-589. GRABNER, M., FRIEDRICH, K., KNAUS, H.-G., STRIESSNIG, J., SCHEFFAUER, F., STAUDINGER, R., KOCH, W.J., SCHWARTZ, A. & GLOSSMANN, H. (1991). Calium hannels from Cyprinus arpio skeletal musle. Pro. Nati. Aad. Si. U.S.A., 88, 727-731.

754 S. Berjukow et al Endogenous alium hannels in HEK293 ells GRABNER, M., WANG, Z., HERING, S., STRIESSNIG, J. & GLOSS- MANN, H. (1996). Transfer of 1,4-dihydropyridine sensitivity from L-type to lass A (BI) alium hannels. Neuron, 16, 27-218. HAMILL, O.P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F.J. (1981). Improved path-lamp tehniques for high-resolution reording from ell and ell-free memrane pathes. Pflagers Arh., 391, 85-1. JANIS, R.A., SILVER, P.J. & TRIGGLE, D.J. (1987). Drug ation and ellular alium regulation. Adv. Drug Res., 16, 39-591. KAMP, T.J., PEREZ-GARCIA, M.T. & MARBAN, E. (1995). Coexpression of 1 suunit with L-type alium hannel a,, suunit in HEK293 ells inreases ioni and gating urrents. Biophys. J., 68, A349. LACERDA, A.E., PEREZ-REYES, E., WEI, X., CASTELLANO, A. & BROWN, A.M. (1994). T-type and N-type alium hannels of Xenopus ooytes: evidene for speifi interations with 13 suunits. Biophys. J., 68, 1833-1843. MIKAMI, A., IMOTO, K., TANABE, T., NIIDOME, T., MORI, Y., TAKESHIMA, H., NARUMIYA, S. & NUMA, S. (1989). Primary struture and funtional expression of the ardia dihydropyridine-sensitive alium hannel. Nature, 34, 23-233. PEREZ-GARCIA, M.T., KAMP, T.J. & MARBAN, E. (1995). Funtional properties of ardia L-type alium hannels transiently expressed in HEK293 ells. J. Gen. Physiol., 15, 289-36. RUTH, P., ROHRKASTEN, A., BIEL, M., BOSSE, E., REGULLA, S., MEYER, H.E., FLOCKERZI, V. & HOFMANN, F. (1989). Primary struture of the fl-suunit of the DHP-sensitive alium hannel from skeletal musle. Siene, 245, 1115-1118. SAVCHENKO, A., GLOSSMANN, H. & HERING, S. (1995). Improved miro-perfusion hamer for multiple and rapid solution exhange in adherent single ells. Pflagers Arh., 429, 436-442. SCHREIECK, J., ZONG, X.G., MEHRCE, G., FLOCKERZI, V., SCHMITT, C. & HOFFMANN, F. (1995). Transiently and staly expressed alium hannel suunit in HEK293 ells. Naunyn- Shmied. Arh. Pharmaol., 351, 376. SKRYMA, R., PREVARSKAYA, N., VACHER, P. & DUFY, B. (1994). Voltage dependent Ca2 + hannels in Chinese hamster ovary (CHO) ells. FEBS Lett., 349, 289-294. SOONG, T.W., STEA, A., HODSON, C.D., DUBEL, S.J., VINCENT, S.R. & SNUTCH, T.P. (1993). Struture and funtional expression of a memer of low voltage-ativated alium hannel family. Siene, 26, 1133-1135. WANG, Z., GRABNER, M., BERJUKOW, S., SAVCHENKO, A., GLOSSMANN, H. & HERING, S. (1995). Chimeri L-type alium hannels expressed in Xenopus laevis ooytes reveal role of repeats III and IV in ativation gating. J. Physiol., 486, 131-137. WILLIAMS, M.E., MARUBIO, L.M., DEAL, C.R., HANS, M., BRUST, P.F., PHILIPSON, L.H., MILLER, R.J., JOHNSON, E.C., HARPOLD, M.M. & ELLIS, S.B. (1994). Struture and funtional haraterization of neuronal ale alium hannel sutypes. J. Biol. Chem., 269, 22347-22357. (Reeived Otoer 18, 1995 Revised January 17, 1996 Aepted Feruary 9, 1996)