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Cell Calcium 42 (2007) 477–487

Ca2+ channel currents and contraction in CaV�3-deficientileum smooth muscle from mouse

Brigitte Held a,∗, Volodymyr Tsvilovskyy a, Marcel Meissner a, Lars Kaestner b,Andreas Ludwig c, Sven Mossmang d, Peter Lipp b, Marc Freichel a, Veit Flockerzi a

a Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Medizinische Fakultat,Universitat des Saarlandes, D-66421 Homburg, Germany

b Institut fur Anatomie und Zellbiologie, Medizinische Fakultat, Universitat des Saarlandes, D-66421 Homburg, Germanyc Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie, Universitat Erlangen-Nurnberg,

Fahrstr. 17, D-91054 Erlangen, Germanyd Institut fur Pharmakologie und Toxikologie, Technische Universitat, Biedersteiner Str. 29, D-80802 Munchen, Germany

Available online 18 June 2007

bstract

Voltage activated L-type Ca2+ channels are the principal Ca2+ channels in intestinal smooth muscle cells. They comprise the ion conductingaV1 pore and the ancillary subunits �2� and �. Of the four CaV� subunits CaV�3 is assumed to be the relevant CaV� protein in smooth muscle.

n protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the CaV1.2, CaV�2, CaV�2 and CaV�3 proteins,ut not the CaV�1 and CaV�4 proteins. Protein levels of CaV1.2, CaV�2 and CaV�2 are not altered in ileum smooth muscle obtained fromaV�3-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the CaV�2 nor thether CaV� proteins appear to substitute for the lacking CaV�3. L-type Ca2+ channel properties including current density, inactivation kineticss well as Cd2+- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence ofCaV�3 protein. However, a key hallmark of the CaV� modulation of Ca2+ current, the hyperpolarisation of channel activation is slightly but

ignificantly reduced by 4 mV. In addition to L-type Ca2+ currents T-type Ca2+ currents could be recorded in the murine ileum smooth muscleells, but T-type currents were not affected by the lack of CaV�3. Both proteins, CaV�2 and CaV�3 are localized near the plasma membrane andhe localization of CaV�2 is not altered in CaV�3 deficient cells. Spontaneous contractions and potassium and carbachol induced contractions

re not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that inleum smooth muscle cells, CaV�3 has only subtle effects on L-type Ca2+ currents, appears not to be required for spontaneous and potassiumnduced contraction but might have a function beyond being a Ca2+ channel subunit.

2007 Published by Elsevier Ltd.

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eywords: Ileum smooth muscle; Voltage activated Ca2+ channel; CaV�3;

. Introduction

Voltage activated Ca2+ channels convert depolarization ofhe plasma membrane to an influx of Ca2+ ions that initiates

uscle contraction, secretion, neurotransmission and genexpression. They have a pore forming �1 subunit, whichogether with the ion conducting pores of voltage-activated

∗ Corresponding author at: Institut fur Experimentelle und Klinische Phar-akologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg,ermany. Tel.: +49 6841 16 26411; fax: +49 6841 16 26402.

E-mail address: brigitte.held@uks.eu (B. Held).

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143-4160/$ – see front matter © 2007 Published by Elsevier Ltd.oi:10.1016/j.ceca.2007.04.013

tion measurements

odium channels (NaV) belong to the family of four domainhannel proteins. Eleven genes encode these CaV�1 subunitshat fall into three distinct subfamilies, CaV1, CaV2 andaV3; in addition, a single novel CaV-like protein has been

dentified that defines a fourth subfamily [1]. The CaV3hannels conduct low voltage activated or T-type Ca2+

urrents, whereas CaV2 and CaV1 channels conduct higholtage activated currents. Among the high voltage activated

hannels CaV2 channels are responsible for N-, P/Q- and-type Ca2+ currents, which are primarily detectable ineurones. The CaV1 channel subfamily comprises four mem-ers, CaV1.1, CaV1.2, CaV1.3 and CaV1.4, which underly

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78 B. Held et al. / Cell C

-type Ca2+ currents. The CaV1 and CaV2 channels areomplexes of the pore forming �1 subunit of ∼190–230 kDassociated with auxiliary � subunits (CaV�), �2� subunitsnd in some cases a � subunit.

Molecular cloning has identified four CaV� subunitsnd a comparison of the distribution of CaV� and CaV�1ubunits indicates that there is certainly not an exclusivessociation between particular pairs of CaV�1 and CaV�ubunits [2,3]. The CaV� subunits bind to the main poreorming CaV�1 subunits and as ancillary cytoplasmic pro-eins modulate the Ca2+ channel function [4]. Among otherffects they shift the voltage dependence of activation in theyperpolarizing direction, so that the channels open at lessepolarized potentials and they are also required to enhancehe number of functional channels at the plasma mem-rane. In addition, CaV� subunits may mediate the inhibitorynd facilitating actions of Ras-related GTPases includingem, Rem2, Rad and GemKir [5–7] and Ca2+/calmodulinependent protein kinase II on L-type Ca2+ currents8].

Pancreatic � cells express mainly CaV�2 and CaV�3 sub-nits and mice deficient in the CaV�3 subunit showed aore efficient glucose homeostasis compared to wild typeice [9]. This is accomplished by an increase of glucose-

timulated insulin secretion whereas the number of L-typea2+ channels in the plasma membrane and the biophysicalroperties of their currents are not affected [9]. Apparently,he CaV�2 subunit is sufficient in maintaining the func-ion and number of L-type Ca2+ channels in the plasma

embrane of these cells. These results indicate that theaV�3 subunit protein may have functions beyond beingsubunit of voltage activated Ca2+ channels which could

nclude the negative modulation of the formation of inos-tol 1,4,5-trisphosphate [9]. The molecular targets of thisdditional CaV�3 function are not known but recently theariant of another CaV� subunit, CaV�4, has been shownegulating gene transcription [10]. Like the CaV�3 func-ion in pancreatic � cells this function of the CaV�4 variantppears to be independent of its function as a Ca2+ channelubunit.

Here we studied the role of CaV�3 in another primary cellystem, in acutely isolated ileum smooth muscle cells. CaV�3as been shown to be expressed in aorta, trachea and lung11,12] and is considered the predominant CaV� subunit ofmooth muscle voltage activated Ca2+ channels. The princi-al expressed �1 subunit in smooth muscle is encoded by theaV1.2 gene [13] and smooth muscle-specific inactivationf this gene yielded mice which suffer from reduced faecesxcretion, absence of rhythmic contractions in small and largentestinal muscle and signs of paralytic ileus [13]. We nowhow that CaV�2 and CaV�3 but not CaV�1 and CaV�4 areeadily detectable in isolated cells from the ileum longitudi-

al smooth muscle layer. Deletion of the CaV�3 gene doesot influence expression levels of the protein subunits of the-type Ca2+ channel CaV1.2, �2-1 and CaV�2. In addition,-type Ca2+ currents and depolarization- and carbachol-

mpts

42 (2007) 477–487

nduced contractions were almost identical in ileum smoothuscle cells and ileum longitudinal smooth muscle strips

rom CaV�3-deficient and wild type animals. Apparently, inleum smooth muscle like in pancreatic islets, CaV�3 mayerve functions beyond its function as an L-type Ca2+ channelubunit.

. Materials and methods

.1. Cell preparation

Smooth muscle cells were acutely isolated from wildype (129SvJ) and CaV�3-deficient (129SvJ background, 10enerations back-crossed) adult mice of either sex. Miceere sacrificed by cervical dislocation according to localuidelines, the abdomen was opened and the small intes-ine exposed. The longitudinal muscle layer 3–4 cm prioro the caecum was carefully stripped off, and the pieceslaced in warmed solution consisting of (in mM): 120 NaCl,KCl, 2 CaCl2, 1.2 MgCl2, 12 glucose, 10 HEPES, pH 7.4.arger muscle strips were cut into smaller pieces, brieflyashed with a Ca2+ and Mg2+-free solution (same solu-

ion as before, CaCl2 and MgCl2 omitted) and placed for0 min into a Ca2+ and Mg2+-free digestion solution con-aining 1 mg/ml collagenase type 1A, 1 mg/ml bovine serumlbumin (BSA) and 1 mg/ml soy-bean trypsin inhibitor (alleagents from Sigma, Germany) at 37 ◦C. Muscle pieces wereashed at least five times with Ca2+ and Mg2+-free solution

o remove the enzymes and triturated approximately 20–30imes with polished Pasteur-pipettes to release single cellsrom the tissue. The volume was increased, equivolume ofa2+ and Mg2+-free and Ca2+ and Mg2+-containing solu-

ion, and the cell suspension stored at 4 ◦C until use theame day.

.2. Electrophysiology

For patch-clamp recordings, ileal smooth muscle cellsere plated onto glass coverslips, after 10 min to allow cells

o adhere to the glass, cells were washed with bath solutionontaining (in mM): 145 tetraethylammonium (TEA) chlo-ide, 10 BaCl2, 1 MgCl2, 10 glucose and 10 HEPES, pH.4. Whole-cell patch-clamp experiments [14] were carriedut at room temperature. The patch pipette solution con-ained (in mM): 145 Cs-aspartate, 5 MgCl2, 5 Mg-ATP, 20GTA, 10 HEPES, pH 7.2. Currents were activated by stepepolarisation every 5 s from a holding potential of −90 or60 mV, between −70 and +70 mV for 400 ms in 10 mV

ncrements to obtain current–voltage (IV) relationships. An0 ms depolarisation to −40 mV was applied prior to the testulse to inactivate T-type channels, which were observed in

ost cells. To measure steady-state inactivation, a 5 s pre-

ulse to potentials from −120 to +20 mV were applied, thenhe pulse (−40 mV, 80 ms) to inactivate T-type currents andubsequently the test pulse to 0 mV for 300 ms. In some cells,

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a2+ channel currents were also activated every 5 s by rampepolarisation from −110 to +60 mV for 170 ms. A P/4 pro-ocol with a leak holding potential of −120 mV was usedor linear leak and capacitance subtraction. Currents wereecorded with an EPC9 patch-clamp amplifier (HEKA, Ger-any), filtered at 1.5 kHz, digitised at 5 kHz. Cell capacitance

nd series resistance were cancelled electronically prior toach pulse, a 10 mV liquid junction potential was applied toll voltages. Peak currents were measured, each IV curve wastted with a modified Boltzman equation of the form I = gmaxV − Erev)/(1 + exp (−V − V1/2)/k) [15] with gmax being theaximal whole-cell conductance, Erev the apparent rever-

al potential, V1/2 the voltage for half-maximal activationnd k the slope factor. Individual steady-state inactivationurves were fitted with a Boltzman equation of the form/Imax = 1/(1 + exp((V − V1/2)/k), with V1/2 being the voltageor half-inactivation and k the steepness factor. To determinehe inactivation rate, the peak L-type current was taken for00%, the current at the end of the 400 ms depolarisation waseasured and calculated as percentage of the peak current. In

ome experiments, Ca2+ channels were repeatedly activatedy step depolarisation from −90 to 0 mV or voltage ramps asescribed above, after approximately 60–70 s, until the cur-ent amplitude stabilised, Cd2+ (300 �M) or (−)-BayK 86445 �M) were applied to the cell by low pressure ejection. Allxperiments were carried out at room temperature. Data areiven as mean ± S.E.M., statistical significance was testedith a Student’s t-test, paired or unpaired as appropriate. Cells

rom wild type and CaV�3-deficient mice were indistinguish-ble, there was no obvious difference in the cell morphologyr membrane capacitance (wild type: 44.2 ± 1.5 pF (n = 76),aV�3−/−: 43.2 ± 1.4 pF (n = 65)).

.3. Contraction measurements

Longitudinal muscle layers from adult (2–6 months) wildype (129SvJ) or CaV�3-deficient mice were prepared byarefully removing ca. 1 cm long strips 4–5 cm prior tohe caecum, and tying silk thread to each end to hang theuscle strip vertically into an organ bath (Fohr FMI, Ger-any). The tissue was placed into a 5 ml organ bath withrebs-Henseleit solution containing (in mM): 118 NaCl, 24.9aHCO3, 4.7 KCl, 2.52 CaCl2, 1.64 MgCl2, 1.18 KH2PO4,.56 glucose, 1 Na-pyruvate, bubbled with carbogen (95%2, 5% CO2). After a resting tension of 0.5 g was applied to

ach muscle strip, an equilibration period of approximately5–60 min with occasionally washing steps was allowed.

modified Krebs–Henseleit solution with 80 mM KCl (Naoncentration reduced accordingly) was applied 3–5 times,he peak in tension was measured, values from the multiplepplications were averaged and taken as 100% contractionor that muscle strip. Concentration–response curves were

btained twice with KCl (40, 60, 80, 100, 120 mM), andumulatives concentration–response curves were obtainedhree times with TEA (0.3, 1, 3, 10, 30 mM) and carbacholCCh, 0.01, 0.03, 0.1, 0.3, 1, 3 �M). Towards the end of

sops

42 (2007) 477–487 479

he experiment, nifedipine (10 �M) was added for 10 minrior to the application of 80 mM KCl. After several wash-ng steps, 10 �M nifedipine was added again for 10 min andhe effect of 3 �M CCh was tested. Tension was averagedver a stable period with a brief delay after the application,ultiple values were averaged and calculated as percentage

f the peak tension obtained from the 80 mM KCl addition.etween applications, an equilibrium time of 10–15 min wasiven.

.4. Western blots and immunocytochemistry

Smooth muscle cells from the ileum longitudinal layerrom wild type and CaV�3−/− mice were collected and lysedn 160 �l of lysis buffer containing (in mM): 50 Tris–HCl, pH.0, 150 NaCl, 5 EGTA, 1% NP40, 0.5% Na-deoxycholate,.1% SDS, 0.0001 phenylmethanesulfonylfluoride (PMSF).he protein lysates (50 �g per lane) were separated on 10%DS-PAGE, and proteins were blotted and probed with thenti-�2�-1 (MAb 20A Alexis Biochemicals) antibody andhe anti CaV1.2 antibody. Blots were stripped and reprobedith the anti-CaV� subunit antibodies [3,9,12,16].Smooth muscle cells were isolated as described above but

eld in a solution containing 0.6 mM Ca2+ and 0.4 mM Mg2+

o prevent strong myocyte contraction. Cells were plated ontooly-L-lysine coated glass coverslips, once washed with PBSnd fixed with 4% paraformaldehyde for 10 min at room tem-erature. Subsequently, cells were washed and permeabilisedith 0.05% Triton X-100 in PBS for 60 s. Following anotherashing step, the cells were incubated for 1 h at room temper-

ture with blocking solution (5% BSA/TBS, 0.25% NaN3)nd then overnight at 4 ◦C with the primary anti-CaV�3-ntibody 828 (1:150 dilution, in 1% BSA/TBS, 0.05% NaN3)r the anti-CaV�2-antibody 425 [16] at a 1:5000 dilution,n 1% BSA/TBS, 0.05% NaN3. Cells were washed 3× formin, incubated with the secondary goat anti-rabbit IgG con-

ugated to AlexaFluor594 (Molecular Probes, 1:1000 dilutionn 5% BSA/TBS, 0.25% NaN3) at room temperature for 1 h,ashed 3× and mounted in 75% glycerol/TBS onto micro-

cope slides. As negative control, the primary antibody wasmitted. Immunofluorscence was recorded using a Nipkow-isc based laser-scanning confocal microscope comprisingconfocal head (QLC-100, VisiTech Int., UK) attached

o an upright microscope (Eclipse E600, NIKON, Japan).he fluorescence was detected by CCD-cameras (Orca ER,amamatsu, Japan). Confocal images were obtained by exci-

ation of the specimen at 561 nm (solid state laser, Mellesriot, Germany) and recording of the emitted light at wave-

engths >570 nm running VoxelScan Software (VisiTech Int.,K). Recording parameters such as camera binning, expo-

ure time and excitation energy were kept constant betweenll experiments. The raw images were processed in ImageJ

oftware (Wayne Rasband, NIH, Bethesda, USA). Z-stacksf the labelled smooth muscle cells were recorded with aeizo-controlled z-stepper (PI, Berlin, Germany) with a stepize of 0.2 �m.

480 B. Held et al. / Cell Calcium

Fig. 1. Western blots of protein extracts (50 �g per lane) from wild type(+) and CaV�3-deficient (−) mouse ileum longitudinal smooth muscle cellsusing CaV1.2-, �2-1, CaV�2- and CaV�3-specific antibodies. The proteinswere run on a single 10% SDS-polyacrylamide gel, blotted and probed withthe CaV1.2- and �2-1- antibodies (left two panels). These blots were strippedand reprobed with the CaV�2- and CaV�3-specific antibodies (right two pan-erp

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. Results

.1. Expression of CaVβ2 and CaVβ3 in ileal smoothuscle

When we studied expression of the subunits of L-typea2+ channels in protein lysates obtained from ileum lon-itudinal smooth muscle from wild type mice by Westernlot we noticed that the CaV1.2 and CaV�2 proteins wereeadily detectable (Fig. 1, left lanes, +) as well as CaV�2nd CaV�3 but not CaV�1 or CaV�4. Both CaV� subunits,aV�2 and CaV�3 might be associated with the CaV1.2 pro-

ein and in CaV�3-deficient cells CaV�2 may substitute forhe lack of CaV�3. In CaV�3-deficient cells CaV�3 is noonger detectable (Fig. 1, CaV�3, right lane, −) but we foundo significant differences between wild type and CaV�3-eficient smooth muscle in the expression levels of CaV�2,aV1.2 and CaV�2 (Fig. 1, right lanes, −). In addition CaV�1nd CaV�4 were not detectable in CaV�3-deficient cells as inild type cells. These findings suggest that there is no appar-

nt compensatory increase of the expression of the other Ca2+

hannel subunit proteins caused by the lack of CaV�3 pro-ein expression. In the following we studied whether the lackf CaV�3 affects L-type Ca2+ currents, their pharmacologynd the contractile properties of longitudinal ileum smoothuscle strips.

.2. L-type Ca2+ channel properties

Single longitudinal myocytes were acutely isolated anda2+ channel currents recorded with Ba2+ as charge carrier

IBa). In response to voltage ramps from −110 to +60 mV, inost cells, a shoulder at more negative and a peak at more

ositive potentials were observed. To test the occurrence of-type Ca2+ channel currents in this preparation, a relativeegative holding potential of −90 mV was used in additiono −60 mV as the physiological resting potential of these

mac

42 (2007) 477–487

ells is approximately −50 to −60 mV [17]. A typical cur-ent response with a shoulder at approximately −30 mV and aeak at about 0 to +10 mV illustrated in Fig. 2A, indicating theresence of both T- and L-type Ca2+ channels. To minimise T-ype channels during recording of L-type current IV-curves, aepolarisation to −40 mV for 80 ms was applied prior to theest pulse. During this prepulse, a peak in current amplitude of

ore than −1.2 pA/pF indicated the presence of T-type cur-ents in the majority of cells (wild type: 48/51, CaV�3−/−:3/45, at a holding potential of −90 mV; wild type: 39/41,aV�3−/−: 37/37, at a holding potential of −60 mV). Theveraged T-type current amplitude during the prepulse wasariable but not significantly different between wild typend CaV�3-deficient myocytes (HP −90 mV: wild type:4.5 ± 0.3 pA/pF (n = 48), CaV�3−/−: −4.1 ± 0.3 pA/pF

n = 43); HP −60 mV: wild type: −3.1 ± 0.2 pA/pF (n = 39),aV�3−/−: −2.9 ± 0.3 pA/pF (n = 37)), indicating that the-type Ca2+ channel is not affected by the lack of CaV�3ubunit. Cells with a current smaller than −1.2 pA/pF werexcluded from this analysis to avoid background noise arte-acts. The more depolarised holding potential of −60 mVesulted in a significant reduction of the T-type current (wildype: p < 0.002; CaV�3−/−: p < 0.05) but did not abolish it.

Average current–voltage (IV-) relationships were recordedt a holding potential of −90 mV and at −60 mV to com-are the L-type channel amplitude between wild type andaV�3-deficient ileal myocytes. At a holding potential of90 mV, the current–voltage relationships of the two geno-

ypes are quite similar, statistically significant differencesere only observed at a test potential of −30, −20 and60 mV (indicated as asterisks), as shown in Fig. 2B. The-type Ca2+ channel current (IBa) amplitude at 0 mV inild type cells was −19.9 ± 1.8 pA/pF (n = 47), and inaV�3-deficient myocytes −17.4 ± 2.2 pA/pF (n = 44). Rep-

esentative current traces are shown in Fig. 2C. The currentn the CaV�3-deficient cells is somewhat but statistically notignificantly smaller than in wild type myocytes. Similarly,here was slight reduction in the L-type Ca2+ current ampli-ude in CaV�3-deficient cells when the holding potential wasepolarised to −60 mV, illustrated in Fig. 2E and F. At 0 mV,he amplitude in wild type myocytes was −25.0 ± 2.1 pA/pFn = 38), and −20.0 ± 2.4 pA/pF (n = 36) in cells lacking theaV�3 subunit, illustrated by representative current traces inig. 2F.

The recorded L-type current amplitude was found to beighly variable. The box plot in Fig. 2D summarises thisariability in the current amplitude at 0 mV evoked from aolding potential of −90 or −60 mV, with the 25th and 75thercentile, the whiskers indicate the 5–95% range. The min-mal and maximal amplitude is indicated as x showing thathe variable amplitude in these cells occurs; however, in bothenotypes, the mean is shown as an open square.

Individual IV relationships were fitted with a Boltz-ann equation (see Section 2) to obtain further information

bout the L-type current properties. The maximal whole-ell conductance was not statistically different between

B. Held et al. / Cell Calcium 42 (2007) 477–487 481

Fig. 2. (A) Representative current traces (IBa) of T- and L-type Ca2+ channels during a voltage ramp from −110 to +60 mV (holding potential: −90 mV; top:voltage protocol) in a wild type (black) and a CaV�3-deficient cell (grey). (B) Averaged current–voltage relationship of L-type Ca2+ channel currents at aholding potential of −90 mV in wild type (closed circles, n = 47) and CaV�3-deficient myocytes (open circles, n = 44). Asterisks indicate statistical significantdifferences (p < 0.05) between wild type and CaV�3−/− curves. (C) Representative current traces of wild type and CaV�3−/− cells at test potentials from−60 to + 50 mV from a holding potential of −90 mV, a prepulse to −40 mV for 80 ms preceded the test pulse to inactivated T-type currents; voltage protocol attop. Scale bars: 5 pA/pF, 100 ms. (D) Box plot summary of current densities at 0 mV of wild type and CaV�3−/− at a holding potential of −90 mV (left, wildtype, n = 47, CaV�3−/−, n = 44) and −60 mV (right wild type, n = 38, CaV�3−/−, n = 36). Mean value is indicated by square, box indicates the 25th and the75th percentile with median, whiskers indicate range from 5 to 95%, indicate highest and lowest measured value. (E) Averaged current–voltage relationshipof L-type Ca2+ channel currents (I ) in wild type (closed circles, n = 38) and Ca � −/− cells (open circles, n = 36) from a holding potential of −60 mV.A type anp otocolc

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ild type and CaV�3-deficient smooth muscle cells. Atholding potential of −90 mV, it was 0.34 ± 0.03 pS/pF,= 47 in wild type and 0.32 ± 0.04, n = 44 (p > 0.05) inaV�3-deficient cells. At the holding potential of −60 mV,

he maximal whole-cell conductance obtained in CaV�3-eficient myocytes was 0.35 ± 0.04, n = 36, compared to.42 ± 0.03, n = 38 (p > 0.05) in wild type myocytes. How-ver, the voltage for half-activation V1/2 and the slopeactor k were different for the L-type current when evokedrom −90 mV (V1/2: wild type, −20.2 ± 0.9 mV, n = 47,aV�3−/−, −16.2 ± 1.0 mV, n = 44, p < 0.005; k: wild

ype, 5.6 ± 0.4 mV, n = 47, CaV�3−/−, 7.6 ± 0.6 mV, n = 44,< 0.005). In contrast to V1/2 of the L-type current obtainedt a holding potential −90 mV, there was no change at aolding potential of −60 mV (wild type: −19.0 ± 0.8 mVn = 38); CaV�3−/−: −18.0 ± 0.9 mV (n = 36)). Only thelope factor k is statistically significantly different at a hold-ng potential of −60 mV (wild type, 4.7 ± 0.3 mV (n = 38),aV�3−/−, 6.2 ± 0.4 mV (n = 36), p < 0.005). Nor was there,t either holding potential, a significant difference in thepparent reversal potential Erev observed (−90 mV: wild type,7.5 ± 1.2 mV, n = 26, CaV�3−/−, 54.0 ± 1.8 mV, n = 35;

60 mV: wild type, 56.5 ± 1.4 mV, n = 24, CaV�3−/−,

8.1 ± 1.2 mV, n = 25). These results are in agreement withhe observation that the gross anatomy of the intestines isot altered. Moreover, no morphological/anatomical changes

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d CaV�3−/− IV-curves. (F) Representative current traces from a holdingin 2.C, except for holding potential) in wild type (top) and CaV�3-deficient

r symptoms regarding the gastro-intestinal tract have beeneported in mice lacking the CaV�3 subunit of the voltage-ependent Ca2+channel [19,20].

To test whether the current recorded in the myocytes isntirely caused by voltage-dependent Ca2+ channels we usedhe non-specific Ca2+ channel blocker Cd2+ at a concentrationf 300 �M, when voltage-ramps from −110 to +60 mV werepplied from a holding potential of−90 mV. Addition of Cd2+

fter approximately 60 s abolished the Ca2+ channel currentn both wild type and CaV�3-deficient ileal smooth muscleells as illustrated in Fig. 3A. In wild type cells, the peak-type current amplitude was decreased from −15.6 ± 2.6 to0.87 ± 0.25 pA/pF (n = 6), which is a reduction by 93.1%

Fig. 3A, inset). Similarly, in CaV�3-deficient myocytes, theeak L-type current diminished by 91.7% (Fig. 3A, inset)rom −16.3 ± 3.8 to −1.2 ± 0.3 pA/pF (n = 7) in the presencef Cd2+. These data suggest that there is no difference in theensitivity to Cd2+ between ileal smooth muscle cells isolatedrom wild type and CaV�3-deficient mice.

Next, we investigated the sensitivity to the L-type Ca2+

hannel agonist (−)-BayK 8644. Co-expression of the skele-al muscle CaV� subunit isoform with the smooth muscle

aV1.2 had resulted in increasing dihydropyridine binding

21], but was not observed when co-expressing the cardiacaV1.2 with CaV�3 [22]. Again, voltage ramps from −110

o +60 mV were applied and after approximately 60–70 s of

482 B. Held et al. / Cell Calcium 42 (2007) 477–487

Fig. 3. Time course of peak L-type Ca2+ channel current (IBa) during voltage ramps from −110 to +60 mV from a holding potential of −90 mV, prior to andd closedB ). Inseti 7 (A), n

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uring the application of 300 �M Cd2+ (A) (indicated by bar) in wild type (ayK 8644 (wild type: closed circles, n = 11, CaV�3−/−: open circles, n = 7

n wild type (shaded, n = 6 (A), n = 11 (B)) and CaV�3−/−cells (white, n =

ontrol recording 5 �M (−)-BayK 8644 was added to the cell.he peak current in wild type myocytes was increased from20.6 ± 3.2 pA/pF prior to the application of (−)-BayK 8644

o −40.6 ± 5.4 pA/pF (n = 14) in the presence of the ago-ist, and in CaV�3-deficient cells the current was enhanced

rom −23.6 ± 3.3 to −42.8 ± 5.4 pA/pF (n = 7) as illustratedn Fig. 3B. The percentage of peak current enhancementFig. 3B, inset) was larger in wild type (120.0 ± 21.2%) thann CaV�3-deficient cells (86.4 ± 17.7%; Fig. 3B. inset) but

csnv

ig. 4. (A and B) Averaged steady-state inactivation and activation curves at a hoormalised to −120 mV prepulse potential and plotted as a function of the respectivor −60 mV) and CaV�3−/− cells (open circles, n = 30 for −90 mV, n = 33 for −6lotted as a function of the respective test potential for wild type (black square, n= 44 for −90 mV, n = 36 for −60 mV); data are fitted with a Boltzman function (wuring the 300 ms depolaristion to 0 mV at a holding potential of −90 mV (C) or −6olding potential) and in CaV�3−/− cells (open circles, n = 44 at −90 mV, n = 36 atrom the beginning of the step depolaristion to 0 mV, holding potential was −90 mVt −60 mV holding potential) and in CaV�3−/− cells (open circles, n = 44 at −90 m

circles, n = 6) and CaV�3−/− myocytes (open circles, n = 7), or 5 �M (−)-s: bar graph of percentage of Cd2+ block and (−)-BayK 8644 augmentation= 7 (B)).

here was no statistically significant difference between thewo groups indicating that there is no alteration in the sensi-ivity to dihydropyridines.

The steady-state inactivation has been shown previouslyn heterologous expression studies [22–25] and also in native

ells [16,19] to be affected by CaV� subunits. Therefore,teady-state inactivation was measured in ileal longitudi-al smooth muscle cells lacking the CaV�3 subunit of theoltage-dependent Ca2+ channel in comparison to wild type.

lding potential of −90 mV (A) or −60 mV (B), inactivation curves weree prepulse potential for wild type (closed circles, n = 35 for −90 mV, n = 290 mV); activation curves were normalised to a test potential of 0 mV and

= 47 for −90 mV, n = 38 for −60 mV) and CaV�3−/− cells (open square,ild type: black, CaV�3−/−: grey). (C and D) Inactivation rate of L-type IBa

0 mV (D) in wild type (closed circles, n = 47 at −90 mV, n = 38 at −60 mV−60 mV holding potential). (E and F) Time to peak of L-type IBa measured

(E) or −60 mV (F) in wild type (closed circles, n = 47 at −90 mV, n = 38V, n = 36 at −60 mV holding potential), *p < 0.05, **p < 0.02.

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s shown in Fig. 4A, there was no effect on the steady-statenactivation curve at a holding potential of −90 (Fig. 4A)r −60 mV (Fig. 4B). Neither the half maximal potential fornactivation V1/2, nor the slope factor k was not found to be dif-erent (at a holding potential of −90 mV: wild type (n = 35),1/2: −47.1 ± 1.4 mV, k: 19.4 ± 0.6 mV; CaV�3−/− (n = 30),1/2: −46.3 ± 1.8 mV, k: 20.3 ± 0.5 mV; at a holding poten-

ial of −60 mV: wild type (n = 29), V1/2: −42.7 ± 1.4 mV, k:7.1 ± 0.8 mV, CaV�3−/− (n = 33): V1/2: −42.1 ± 1.7 mV,: 16.7 ± 0.8 mV). These data imply that the CaV�3 subunitlays no role for the steady-state inactivation behaviour of L-ype Ca2+ channels in these smooth muscle cells from mouseleum.

Similarly to inactivation, activation has been reported toe altered by the presence or absence of CaV� subunits16,19,26]. As with steady-state inactivation, in smooth mus-le cells from mouse ileum, there was no difference inctivation when comparing wild type and CaV�3-deficientells (Fig. 4A and B). At a holding potential of −60 mV,he midpoint of activation of the normalised IV curves was

20.7 ± 0.8 mV in wild type (n = 38) and −20.4 ± 0.8 mVaV�3-deficient cells (n = 36). At the more negative hold-

ng potential of −90 mV, the midpoint of activation was22.3 ± 0.8 mV and −20.9 ± 1.0 mV for wild type (n = 47)

nd CaV�3-deficient cells (n = 44), respectively. Although notignificantly different, it goes in the same direction as the V1/2etermined by Boltzman fits to the IV curves (Fig. 2B and).

Moreover, the inactivation rate of the L-type current dur-ng the 400 ms test pulse was also not statistically differentetween the two genotypes irrespective of the holding poten-ial, as shown in Fig. 4C and D. Data were analysed between

30 mV and +30 mV as the L-type Ca2+ channel current waslearly observable in that test potential window. At a potentialf 0 mV, the inactivation rate is 43.4 ± 1.2% (n = 47, holdingotential −90 mV) or 46.0 ± 1.2% (n = 38, holding potential60 mV) in wild type cells and 39.2 ± 2.2% (n = 44, holding

otential −90 mV) or 43.6 ± 1.6% (n = 36, holding potential60 mV) in CaV�3-deficient myocytes.To further analyse the L-type Ca2+ channel current in these

leal cells, the time until the maximal current was recordedtime to peak; Fig. 4E and F). In both genotypes, the time toeak decreased with increasing test potential. At a holdingotential of −60 mV, there was no statistical significant dif-erence between wild type (n = 38) and CaV�3−/− myocytesn = 36). At −90 mV holding potential, there was a statisti-al difference only at a test potential of +10 mV (wild type:3.1 ± 0.7 ms (n = 47), CaV�3−/−: 18.3 + 0.9 ms (n = 44),< 0.02) and +30 mV (wild type: 10.0 ± 0.9 ms (n = 47),aV�3−/−: 13.0 ± 1.2 ms (n = 44), p < 0.05), suggesting that

he activation time of the L-type current in the knock-out cellss only slightly if at all slowed at these two test potentials. In

ummary, these data indicate that the CaV�3 subunit plays atest a subtle role for the functioning or the biophysical prop-rties of the L-type Ca2+ channel in acutely isolated murineleal smooth muscle cells.

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42 (2007) 477–487 483

.3. Tension recordings

Although no major differences between the L-type Ca2+

hannel currents in wild type and CaV�3-deficient myocytesere observed, the possible influence of the CaV�3 subunit on

mooth muscle layer contractility was examined by isometricension measurements performed on strips of ileum longitu-inal smooth muscle. In both wild type muscle and muscleacking the CaV�3 subunit, the baseline showed small sponta-eous oscillating contractions. Tension prior to application ofCl (averaged over ca. 3 min) in relation to 3–5 applicationsf 80 mM KCl which served as 100% contraction controlpeak tension), was similar in both genotypes (wild type:3.3 ± 1.4% of the peak tension, CaV�3−/−: 12.7 ± 1.0%f peak tension). Contractions activated by addition of 40,0, 80, 100 and 120 mM KCl showed no differences betweenuscle strips from wild type and CaV�3-deficient mice

Fig. 5A, E and I). The contraction evoked by applicationf 80 mM KCl was not statistically significantly differentetween wild type and CaV�3-deficient muscle (Fig. 5I).n both genotypes, KCl rapidly increased the tension, fol-owed by a gradual decrease, spontaneous contractions werebolished during that time, but recovered immediately uponashing of the muscle strips.The oscillating spontaneous contractions in both wild type

nd CaV�3-deficient muscle were completely abolished afterhe application of the L-type Ca2+ channel blocker nifedip-ne (wild type: 7.5 + 7.3%, n = 11, CaV�3−/−: 5.3 ± 0.9%,= 8; Fig. 5B and F) indicating that the Ca2+ influx through-type Ca2+ channels is crucial for the spontaneous ilealmooth muscle contraction. KCl (80 mM) applied 10 minfter the addition of nifedipine, induced only a brief andransient contraction, in wild type muscle to a maximumf 24.4 ± 5.0% (n = 10) and in CaV�3-deficient muscle topeak of 26.5 ± 2.2% (n = 8) as shown in Fig. 5J. In con-

rast, 3 �M carbachol (CCh) slowly increased the tension to2.1 ± 7.0% (n = 11) in wild type and to 41.8 ± 5.2% (n = 8)n CaV�3-deficient muscle (Fig. 5J).

Muscarinic stimulation of visceral smooth musclenduces contractions [17,18], therefore a cumulativeoncentration–response curve was recorded using 0.01, 0.03,.1, 0.3, 1 and 3 �M carbachol (CCh; Fig. 5C, G and K).owever no marked difference was observed, the EC50 inild type muscle was 0.26 �M (n = 12), and 0.19 �M inaV�3-deficient muscle (n = 12). Moreover, the cumulativeoncentration–response curve of the K+ channel blockeretraethylammonium (TEA) at concentrations of 0.3, 1, 3,0 and 30 mM was not different between the two genotypesFig. 5D, H and L).

These data indicated that the �3 subunit of the voltage-ated Ca2+ channel in longitudinal smooth muscle layeras little functional roles in the contraction ability evoked

y high K+-containing external bath solution, K+ channellockade by TEA, or muscarinic stimulation using CCh. Inddition, there was no change in sensitivity to the dihy-ropyridine nifedipine at 10 �M, and the contractile effect

484 B. Held et al. / Cell Calcium 42 (2007) 477–487

Fig. 5. Representative traces of contractions induced in wild type (A–D) and CaV�3-deficient (E–H) ileal smooth muscle strips by cumulative KCl depolarisation(A and E), CCh (C and G), TEA (D and H), concentrations as indicated (KCl, TEA in mM, CCh, nifedipine in �M). Abolishment of spontaneous contractionsby 10 �M nifedipine and subsequent addition of 80 mM KCl (B and F). Cumulative concentration–response curves after application of KCl, (I; wild type n = 8;CaV�3−/− n = 8), CCh (K, line is a sigmoidal fit to data; wild type n = 12, CaV�3−/− n = 12) and TEA (L; wild type n = 8, CaV�3−/− n = 8), wild type:closed circles, CaV�3−/−: open circles. (J) Bar graph showing baseline tension prior to addition of nifedipine (wild type, n = 11, CaV�3−/−, n = 8), responsesto 10 �M nifedipine (wild type, white, n = 10, CaV�3−/−, n = 8), 80 mM KCl in the presence of 10 �M nifedipine (wild type, n = 11, CaV�3−/−, n = 8) and3 �M CCh in the presence of 10 �M nifedipine in wild type (white, n = 11) and CaV�3−/− smooth muscle strips (shaded, n = 8), data is given as percentageof maximal contraction during stimulation with 80 mM KCl.

alcium

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f KCl or CCh after blockade of the L-type channels byifedipine.

.4. Subcellular localisation of CaVβ2 and CaVβ3

rotein in ileum smooth muscle cells

In functional L-type Ca2+ channels the CaV� subunits arettached to cytosolic portions of the CaV1 proteins at theytosolic site of the plasma membrane. Because there is noajor effect on the L-type Ca2+ channel properties in ileal

mooth muscle cells lacking CaV�3 and to confirm this local-sation, single acutely isolated ileal smooth muscle cells weresed to stain the Cavp3 or CaV�2 proteins. As in the West-rn blot shown in Fig. 1 the CaV�-specific antibodies weresed and a fluorescently labelled (AlexaFluor594) secondaryntibody. As seen in the confocal sections of two represen-ative wild type cells in Fig. 6A, the CaV�3 subunit is mostrominently localised at the edge of the smooth muscle cells,.e. at the plasma membrane. As expected, in myocytes lack-ng the CaV�3 subunit, the fluorescence was very weak andcattered throughout the cell (Fig. 6B). Similarly, the CaV�2ubunit was localised at the plasma membrane in wild typeFig. 6C) and CaV�3-deficient cells (Fig. 6D). Ileal cells,hich had not been incubated with the primary antibody,

erved as negative controls (Fig. 6E). Here as anticipated,he fluorescent signal was extremely weak, cells were there-ore extremely difficult to localise by their fluorescent signal.hese data confirm the Western blot analysis that Ca �

V 2nd CaV�3 subunits are expressed in ileal smooth muscleells and indicate that they are primarily localised at thelasma membrane, presumably near or in the Ca2+ channelomplex.

arcb

ig. 6. Confocal sections through single representative ileal smooth muscle cells frubunit was stained with the antibody B30 (1:150) (A and B), with the CaV�2 subo AlexaFluor594 was used as secondary antibody (1:1000). As control, the primaictures.

42 (2007) 477–487 485

. Discussion

Our data show that mouse ileum smooth muscle cells doxpress CaV�2 and CaV�3 proteins in addition to the iononducting pore CaV1.2 and the ancillary subunit CaV�2-. We could not detect the a and b variants of CaV�1 noraV�4 by our antibodies, indicating that these proteins areot expressed in this tissue or that they are expressed at veryow levels which escape the sensitivity of our antibodies.n ileum smooth muscle cells from CaV�3-deficient miceo compensatory increase of CaV�2 protein expression wasetectable indicating that CaV�2 does not simply substituteor CaV�3. In addition protein expression levels of CaV1.2nd CaV�2 are apparently identical in cells from wild typend CaV�3-deficient mice.

In line with the protein expression data we could onlynd subtle differences of functional L-type Ca2+ channelroperties recorded from wild type cells and cells obtainedrom CaV�3-deficient mice. In the current–voltage relation-hip obtained at a holding potential of −90 mV, the wholeell current activation is shifted by ∼4 mV to more depo-arised potentials in CaV�3−/− ileum cells when comparedith wild type. A similar shift in the voltage depen-ence of Ca2+ channel currents was observed in sensoryeurones treated with generic anti-CaV�-antisense oligonu-leotides [27], in CaV�2−/− embryonic cardiomyocytes [16]nd in dorsal root ganglion neurones [19]. Other param-ter of current density, activation and inactivation as well

s channel sensitivity towards Cd2+ and the dihydropy-idine (−)-BayK 8644 were not significantly different inells of both genotypes. Moreover, no differences coulde observed in spontaneous contractions or during contrac-

om wild type (A, C and E) or CaV�3-deficient mice (B and D), the CaV�3

unit with AK425 (1:5000) (C and D); a goat anti-rabbit antibody coupledry antibody was omitted (E). The scale bar in (A) is valid for all confocal

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ions elicited by addition of extracellular K+ or carbacholndicating that CaV�3 does not interfere with these cellularunctions.

A possible explanation of the small effect of CaV�3ene deletion on L-type currents and smooth muscle func-ion could be a distinct localisation of the CaV�3 proteinompared to the CaV�2 protein. In a previous study weave shown that CaV�3 negatively modulates InsP3-induceda2+-release without affecting the function of the L-typea2+ channel in pancreatic � cells [9]. This additional

unction might require a specific intracellular targeting ofaV�3. The immunocytochemical data shown here do not

upport such a conclusion. It appears that CaV�3 likeaV�2 is predominantly localized near or at the plasmaembrane.The CaV�3 subunit is assumed to be the relevant CaV�

ubunit of smooth muscle L-type Ca2+ channels. This isn contrast to dorsal root ganglion neurones, where CaV�3s mainly associated with CaV2.2 channels, which underlie-type Ca2+ currents in these cells [19]. However, there iso evidence that intestinal smooth muscle cells and Ca2+

urrents recorded from these cells are affected in the threeaV2.2-deficient mouse models, which have been estab-

ished independently [20,28,29]. In contrast L-type Ca2+

urrents appear to be essential because gene targeting ofhe underlying ion conducting pore CaV1.2 severely affectsa2+ currents in ileum smooth muscle cells and gut function

13].So in summary, we have shown that both, CaV�3 and

aV�2 proteins are expressed in isolated cells from mouseongitudinal ileum smooth muscle. Deletion of the CaV�3ene does not induce compensatory increase of the proteinevels of CaV�2, CaV1.2 or CaV�2. Except for a small CaV�3ependent hyperpolarisation by ∼4 mV of Ca2+ channel acti-ation, L-type Ca2+ currents recorded from isolated smoothuscle cells and contraction of ileum longitudinal smoothuscle strips are not altered upon CaV� deletion supporting

he idea that CaV�3 might serve additional functions beyondts function as a Ca2+ channel subunit.

cknowledgements

We thank Christine Wesely for expert technical assistance;amona Golzer, Sabine Pelvay and Martin Jung for immu-izing and bleeding rabbits. This work was supported by theeutsche Forschungsgemeinschaft (V.F., M.F.), Fonds derhemischen Industrie (V.F.) and Forschungsausschuss derniversitat des Saarlandes (B.H., M.F., V.F., P.L.).

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