From the Cardiology, Swiss Cardiovascular Center Bern, University Hospital, 3010 Bern, Switzerland, Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0828, and Department of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Koeln, Germany
Received for publication, October 31, 2002
, and in revised form, February 10, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In human myocardium expression of three different -subunits genes (
1-
3) (810) was demonstrated at the mRNA and the protein level (3, 10). In human non-failing (NF)1 left ventricular (LV) mRNA our RT-PCR experiments with degenerated primers complementary to coding sequences with high similarity between
2- and
3-genes generated three amplification products of 650, 503, and 483 bp. The 650- and 503-bp amplification products are sequence-identical to
2 and
3 coding sequences (
2: GenBankTM accession number AF423189
[GenBank]
, nn 97746;
3: GenBankTM accession number X76555
[GenBank]
, nn 135637). The 483-bp amplification product contained a deletion of 20 nucleotides matching exon 6 of the
3-gene (11). At least two different isoforms of the human
2-subunit gene are expressed in the human heart (
2a,
2b) (12, 13) that differ with respect to their short amino termini. Our Northern blot experiments revealed transcripts for
2a-,
2b-, and
3-subunits in human heart tissue; therefore, we assessed quantitative expression of these
-subunits by real-time PCR, which demonstrated substantially different expression levels with
2b >
3 »
2a. To investigate the physiological effects of these
-subunits, we first cloned both the full-length coding sequence of the
3-subunit (
3a) and the isoform containing the deletion of exon 6 (20 nn) (
3trunc) from human heart. Deletion of exon 6 results in truncation of the protein, and, interestingly,
trunc is up-regulated in heart failure. Because of the high homology of the rabbit
2a-subunit to the human
2b we used this subunit together with the two cloned human
3-subunit isoforms to study the functional impact of these
-subunits onto the calcium inward current at the single channel level. Electrophysiological studies were designed to answer the following questions. 1) Do human cardiac
3-subunits functionally coexpress with pore subunits from rabbit heart (Cav1.2a), rabbit smooth muscle (Cav1.2b), and human heart (Cav1.2)? Is there a difference in quality and extent of their modulation when compared with a
2-subunit? 2) Does alternative splicing of the
3-subunits in heart failure explain the increase in single channel activity? These questions were addressed by transient coexpression in cell lines stably expressing CaV1.2 isoforms.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR-based Analysis of -Subunit Gene Expression in Human MyocardiumThe experiment is not depicted in this paper. Degenerated oligonucleotide primers complementary to coding sequences of high similarity between human
2- and
3-subunits were obtained from MWG Biotech (Freising, Germany) (sense, 5'-CTGGAGGAGGACCGGGA(A/G)-3'); antisense, 5'-A(A/G)(A/C)GC(C/T)TT(C/T)TGCATCAT(A/G)TC-3'). RT-PCR (40 cycles at 94, 48, and 72 °C, each 30 s) was performed with reverse-transcribed 0.1 µg of mRNA isolated from NF human LV myocardium in 1x PCR-Mix containing 1.5 mmol/liter MgCl2, (pmol/liter) primers 0.2, dNTP 200, and 2.5 units of Taq polymerase (MBIfermentas, St. Leon Rot, Germany). Amplification products were electrophoresed on 0.8% agarose; 650-, 503-, and 483-bp DNA bands were excised, extracted by QuikSpin columns (Qiagen), subcloned into pTAdvantage (Clontech), amplified, and sequenced.
Ratio of 3a-/
3trunc-subunit expression in human heart specimens was investigated by RT-PCR (40 cycles at 94, 60, and 72 °C, each 30 s) using sense primer 1, 5'-GCGGCTAGTGAAAGAGGGCG-3' (nn 356375, X76555
[GenBank]
) and antisense primer 2, 5'-TGGTTGATGGTGTCAGCGTCC-3' (nn 865845, GenBankTM accession number X76555
[GenBank]
) with 0.1 µg of reverse-transcribed mRNA isolated from NF- and ICM-LV (each n = 7). Amplification products were electrophoresed on 5% PAGE (see Fig. 1C), and densitometric analysis of amplified bands was performed by Quantity One (Bio-Rad).
|
TaqMan assays were performed with the iCycler iQ real-time PCR detection system using primer/fluorescent probe concentrations of 200 nM either in 1x iQ Supermix (2a,
2b,
3), or iQ SYBR Green (cardiac calsequestrin) (all Bio-Rad). Quantification of the
2a and
2b expression was set up with common antisense primer and common fluorescent probe (antisense primer 5, 5'-CGGTCCTCCTCCAGAGATACAT-3' (nn 11089, GenBankTM accession number AF 423189); fluorescent probe, 5'-6FAM-ATGGACGGCTAGTGTAGGAGTCTGCCGA XT p-3' (nn 7952; GenBankTM accession number AF 423189), and isoform-specific sense primers primer 3 (
2a), 5'-GCATCGCCGGCGAGTA-3' (nn 2136, GenBankTM accession number AF423189
[GenBank]
); primer 4 (
2b), 5'-GACAGACGCCTTATAGCTCCTCAA-3' (nn 730, GenBankTM accession number AF285239
[GenBank]
) complementary to the particular short N termini.
3-specific TaqMan assay was set up with sense primer 6, 5'-CCACCTGGAGGAGGACTATG-3'; antisense primer 7, 5'-GCAGCAGGAGGCTGTCAGTA-3'; and fluorescent probe, 5'-6FAM-ACCTGTACCAGCCTCACCGCCAACA q-3' (nn 12231242, 14231404, and 12581282, respectively; GenBankTM accession number XM_006783). Assay conditions for
2a,
2b, and
3 were 95 °C for 3 min, 40 cycles at 95 and 56.5 °C (
3, 60 °C), each 30 s, in 1x iQ Supermix. PCR efficiencies/correlation coefficients for
2a,
2b, and
3 were 99.0%/0.992, 100.5%/0.996, and 104.5%/0.992 respectively. Fluorescent probes were synthesized by TIB MOLBIOL, Berlin, Germany. Cardiac calsequestrin expression was determined using sense primer 8, 5'-AAGGTGGCAGCAAGCAATTC-3' and antisense primer 9, 5'-TTCTCCTGTCCCTGCTAAGTG-3' (nn 13721391 and 15201500, respectively; GenBankTM accession number D55655
[GenBank]
) in 1x iQ SYBR Green (3 min at 95 °C; 40 cycles at 95 and 57.8 °C, each 30 s) with PCR efficiency/correlation coefficient of 99.2%/0.996. Products of amplification were verified by gel electrophoresis and for cardiac calsequestrin additionally by melt curve analysis.
Northern Blot AnalysisFor the 3-subunit 10 µg of mRNA isolated with Trizol (Invitrogen) and poly(A) tract kit (Promega) from human right atrium (RA), right ventricle (RV), and LV obtained from NF/ICM hearts were used for Northern blot analysis. mRNA isolation and Northern blot experiments were performed essentially as described before (14). Northern blots were hybridized at high stringency (16 h at 42 °C in 5x SSC, 1x PE (50 mM Tris-HCl (pH 7.55), 0.1% sodium diphosphate, 1% sodium dodecyl sulfate, 0.2% polyvinylpyrolidone, 0.2% Ficoll, 5 mM EDTA), 50% formamide, with 2 x 106 cpm/ml of a 32P-labeled antisense RNA probe derived from the
3a-subunit-specific 503-bp amplification product (described above) subcloned into pSP72 (specific activity, 1.2 x 108 cpm/µg). NF/ICM Northern blots were washed for 5 min at room temperature in 2x SSC, 0.1% SDS, 10 min in 0.2x SSC, 0.1% SDS, and then at 50 °C in 0.2x SSC, 0.1% SDS (NF, 15 min; ICM, 15 + 10 min). Autoradiography exposure was 96/108 h at 80 °C on Eastman Kodak Co. BioMax MS-1 (Amersham Biosciences) for NF/ICM Northern blots. For the
2-subunit 2 µg of NF LV/RV, ICM LV/RV mRNA were electrophoresed, blotted, and hybridized with isoform-specific 32P-labeled
2a and
2b (nn 7895579474 and 158721159133 respectively; GenBankTM accession number AL390783
[GenBank]
) and C-terminal-specific
2common (nn 13742327, GenBankTM accession number U95019
[GenBank]
). Antisense RNA probes were subcloned into pGEM-3Z (Promega) (
2a), pCR 2.1-TOPO (Invitrogen) (
2b), and pTAdvantage (Clontech) (
2common) (specific activities were 6.7, 6.8, and 6.8 x 108 cpm/µg, respectively). Probes were generated by RT-PCR, using particular primers. Blots were hybridized as described above; wash solution I was 2x SSC, 0.05% SDS; wash solution II was 2x SSC, 0.1% SDS. All blots were washed 5 min at room temperature and 2 x 15 min at 65 °C in wash solution I; blots hybridized with
2b/
2a and
2common were washed 5 min/2 x 15 min at 65 °C in wash solution II. Autoradiography exposures were 2, 4, and 2 h, respectively, at room temperature. For cardiac calsequestrin blot was manufactured and processed as described for
2-subunits but hybridized with a 32P-labeled antisense probe specific for human cardiac calsequestrin (nn 10211210, GenBankTM accession number BC022288
[GenBank]
) (specific radioactivity, 9.5 x 108 cpm/µg). Wash protocol was performed with wash solution I for 5 min at room temperature, 2 x 15 min at 65 °C, and 2 x 15 min wash solution II at 65 °C. Autoradiography exposure was 6 h at room temperature.
Cloning of 3aFull-length
3a-subunit sequence was cloned using two pairs of sequence-specific primers derived from X76555
[GenBank]
. 1st pair, 5'-CCATGTATGACGACTCCTAC-3' and 5'-GTTCCCAGATCTCCTGGCCTTC-3' (nn 3756 and 458437); 2nd pair, 5'-GAAGGCCAGGAGATCTGG-3' and 5'-GGCTGCAGGAGGCTGTCAGTAGCT-3' (nn 437454 and 15081485 (
3a) or 14881465 (
3trunc); 2nd antisense primer contains point a mutation at the 4th position (A
T) to create a PstI restriction site). The
3a full-length clone was constructed using the internal BglII and the generated PstI restriction site and is sequence-identical to X76555
[GenBank]
.
Cell Culture and TransfectionCell culture and transient cotransfection were done as described (15, 16). In brief, Chinese hamster ovary (CHO) cells were stably transfected with the Cav1.2a-subunit cloned from rabbit heart (17) or with the Cav1.2b-subunit from rabbit lung (18). Human embryonic kidney cells (HEK 293) were stably transfected with cDNA encoding the human cardiac Cav1.2-subunit (NM_000719 [GenBank] ) (19). For experiments with pore subunits alone, cells were seeded in polystyrene Petri dishes (9.6 cm2; Falcon, Heidelberg, Germany) at a density between 104 and 2 x 104 cells cm2 and used within 4896 h after plating.
For eukaryotic expression cloned human 3a-,
3trunc-, and rabbit
2a- and
2
-1-coding sequences were subcloned into pcDNA3.1 (Clontech). CHO or HEK 293 cells were transiently cotransfected with the cDNA plasmids encoding the different
-subunits together with
2
-1-subunit (from rabbit skeletal muscle (20)) and green fluorescence protein (pGFP; Clontech). Lipofection was carried out by incubating (36h) with SuperFect (Qiagen) and the respective plasmids at a DNA mass ratio of 3:3:1 (15). Transfected cells were grown on Petri dishes in Dulbecco's modified Eagle's medium (Biochrom KG, Berlin, Germany) supplemented with 10% fetal bovine serum (Sigma), and penicillin (10 units ml1) and streptomycin (10 µg ml1; both Biochrom). Electrophysiological recordings were conducted 4872 h after transfection.
Electrophysiological RecordingsWhole-cell and single channel currents through L-type Ca2+ channels were measured at room temperature (1923 °C). Whole-cell experiments were performed in external solution containing (mmol/liter) NaCl 120, BaCl2 10.8, MgCl2 1, CsCl 5.4, dextrose 10, HEPES 10 (pH 7.4). Pipettes (borosilicate glass, 57 megohms) were filled with (mmol/liter) CsCl 120, MgCl2 3, MgATP 5, EGTA 10, HEPES 5 (pH 7.4). Ba2+ currents were elicited at 0.2 Hz by depolarizing voltage steps from a holding potential of 80mV to various test potentials as indicated. Currents were sampled at 10 kHz and filtered (3 dB) at 2kHz (List EPC-9; HEKA, Lambrecht, Germany). Leak and capacitive currents were subtracted by using a P/N pulse protocol. Peak currents were determined using the average of a 5-ms time window. Time-dependent inactivation was analyzed as I100, the current remaining at the end of a 100-ms test pulse, relative to peak current. The software PULSE/PULSE-FIT (version 9.12; HEKA) was used for data acquisition and analysis.
Single channel measurements and analysis were done as reported (5). Cells were superfused with bath solution containing (mmol/liter) potassium glutamate 120, KCl 25, MgCl2 2, HEPES 10, EGTA 2, CaCl2 1, Na2-ATP 1, dextrose 10 (pH 7.4 with NaOH, 2123 °C). Pipettes (710 megohms) were filled with (mmol/liter) BaCl2 110, HEPES 10 (pH 7.4 with tetraethylammonium hydroxide). Single calcium channels were recorded in the cell-attached configuration (depolarizing test pulses of 150-ms duration at 1.67 Hz, holding potential 100 mV). An Axopatch 1D amplifier and PClamp 5.5 or 6.0 software (both Axon Instruments, Foster City, CA) were used for pulse generation, data acquisition (10 kHz), and filtering (2 kHz, 3 dB, 4-pole Bessel filter). Experiments were analyzed whenever the channel activity persisted for at least 72 s (120 sweeps, using 180 sweeps in most cases). Linear leak and capacity currents were subtracted digitally. Openings and closures were identified by the half-height criterion. The availability (fraction of sweeps containing at least one channel opening), the open probability (popen, defined as the relative occupancy of the open state during active sweeps), and the peak ensemble average current (Ipeak, obtained visually) were analyzed from single channel and multichannel patches. In the latter case, they were corrected for n, the number of channels in the patch. n was defined as the maximum current amplitude observed, divided by the unitary current. Peak current was corrected by division through n. The availability was corrected by the square root method: (1 availabilitycorrected) is the nth root of (1 availabilityuncorrected). The corrected popen was calculated on the basis of the corrected number of active sweeps, i.e. total open time divided by (n x availabilitycorrected x number of test pulses x pulse length). Closed time and first-latency analyses were carried out in patches where n = 1. Time constants of open time and closed time histograms were obtained by maximum-likelihood estimation (PStat software; Union City, CA and Axon Instruments).
Data AnalysisEffects of the -subunits on single channel parameters were statistically examined by one-way ANOVA over the whole series (i.e. untransfected controls of a given pore subunit and cells cotransfected with
2
, pGFP, and pcDNA3.1 or a given
-subunit subcloned into pcDNA3.1). Significant ANOVAs were followed by post-tests against pcDNA3.1 and among
subunits, applying Bonferroni correction for multiple comparisons. p < 0.05 was considered significant. All values are given as mean ± S.E.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Abundance of the 2a-,
2b-, and
3-subunit mRNA expression was determined by real-time RT-PCR in NF LV mRNA (n = 3) and ICM LV mRNA (n = 5; mean echocardiographic ejection fraction of examined ICM hearts, 21 ± 2.23% (S.D.)). Expression data in each specimen were normalized to the respective expression of the housekeeping gene cardiac calsequestrin (3, 21), which was also determined by real-time RT-PCR. Real-time RT-PCR for
2a/
2b was designed with isoform-specific N-terminal sense primers but common C-terminal antisense primer and fluorescent probe. Because we detected no significant differences of copy numbers among
2a,
2b, and
3 when comparing NF LV and ICM LV, data were pooled. In the specimens examined the
2b copy number was 1.49 x 105 ± 6.18 x 104 which is 35 times the
2a copy number of 4.11 x 103 ± 1.42 x 103 (p = 0.01) (Fig. 2C). Real-time RT-PCR for the
3 detected 4.83 x 104 ± 1.48 x 104 copies in the eight specimens examined, which was significantly different from either
2a (p = 0.02) or
2b (p = 0.03) (Fig. 2C). PCR efficiencies of
2a,
2b,
3, and cardiac cardiac calsequestrin were very close to 100%, and correlation coefficients were >0.99, respectively (see "Experimental Procedures"), providing a solid basis for comparison. mRNA copy number was calculated on the basis of a 40% efficiency of reverse transcription (22). Gel electrophoresis of the real time PCR products demonstrated one amplification product of expected size per reaction.
The full-length 3a-sequence cloned from human NF LV mRNA by RT-PCR consists of 1455 bp and encodes 484 amino acids, and this
3 is identical to the
3a-subunit isoform cloned from human thyroidoma (GenBankTM accession number X76555
[GenBank]
). Except for exon 6 deletion (20 nn), which results in a premature stop of translation at nucleotide position 495 no other splicing product of the
3-gene was observed in human heart. A similar truncation was also observed in mouse brain tissue (23). Because truncated proteins may play a role in cardiomyopathy, as demonstrated for troponin I (24), we further investigated the (patho-)physiological relevance of
3trunc by performing RT-PCR experiments using primers 1 + 2 (Fig. 1B) in LV mRNA isolated from NF and ICM (Fig. 1C). In the specimens examined (LV NF/ICM, each n = 7; mean echocardiographic LV ejection fraction in ICM patients, 23.75 ± 2.75% (S.D.)) the ratio of
3a-/
3trunc-subunit isoform expression changed from 83/17% in NF LV to 51/49% in ICM LV.
CHO cells stably expressing the different splice variants of the Cav1.2 L-type calcium channel pore were transiently cotransfected with 2
-subunits together with either rabbit
2a- or the cloned human
3a- or
3trunc-subunits (Fig. 3). The existing rabbit
2a-clone was chosen for our coexpression experiments because of its high homology (96%) to the
2b, which in the human heart is predominantly expressed when compared with human
2a. Overall, cotransfection increased current density, shifted the current-voltage relationship toward more negative potentials, and increased the rate of inactivation, e.g. at the respective potential of peak current density (I100 for non-cotransfected cells, 102 ± 3%;
2a, 66 ± 9%;
3a, 74 ± 4%; and
3trunc, 68 ± 13%; n as in Fig. 3), as reported (12, 25). Although the qualitative effects with cotransfection of rabbit
2a- and human
3-subunits were similar, the effects of
3-subunits, and of
3trunc in particular, were less pronounced. A detailed comparison, and separation of possible effects of cotransfected
2
-subunits, was carried out using the single channel technique.
|
Typical traces from single channel experiments using the recombinant Cav1.2a pore-forming subunit are depicted in Fig. 4. Single channel activity was sparse in patches from non-cotransfected as well from cells cotransfected with 2
-subunits (plus pcDNA3.1 and pGFP) only.
3a and
3trunc increased the probability of finding openings within a test pulse (availability). Open probability within such active sweeps appeared moderately enhanced. In contrast, rabbit
2a cotransfection led to marked increases in both parameters and to a higher increase in the peak current of the ensemble average. These visual impressions were statistically confirmed (Table I). Analysis of fast gating parameters in one-channel patches revealed that the mean closed time is the parameter most prominently affected by rabbit
2a whereas human
3a or
3trunc have no effect on closed times. Histogram analysis showed that the reduction in mean closed times is because of a shortening of the time constant of the slow component
closed,slow. Open time histograms revealed an increase in the time constant of the open state (Table I) but only in the case of rabbit
2a.
|
|
To examine whether the more marked modulation by rabbit 2a-subunits is restricted to the cardiac type Cav1.2a pore subunit, we examined the effect of
2a and
3a on the smooth muscle splice variant, Cav1.2b. As indicated in Fig. 5 and Table II, very similar results were found as with Cav1.2a. Again, both
-subunits increased availability similarly. Ensemble average currents were more markedly affected by
2a because of a significant additional effect on rapid gating and open probability. Similar to Cav1.2a,
closed,slow was reduced, which may explain this effect. Together, these findings confirm a role of the
-subunit structure on pore properties. As with Cav1.2a, non-transfected cells and cells cotransfected only with pcDNA3.1,
2
-subunits, and GFP displayed a quite sparse pattern of activity of Cav1.2b (Fig. 5), similar to our previous observations (26).
|
|
To account for the functional differences found among the multiple splice variants of human cardiac-type Cav1.2 (27, 28), we next examined the Cav1.2 pore-forming subunit usually expressed in human myocardium (19). We expressed cDNAs in HEK 293 cells for these studies, which enabled us to perform a more extended analysis, because of better seal stability. The inherent properties of pore subunits, as well as their differential modulation by rabbit 2a and human
3 isoforms (Table III), were comparable with those of the CHO system. Another common feature with the CHO cell experiments was recognized:
2a but not the
3-subunits increased open probability and open time duration. Closed time duration appeared to be shortened by
2a, mainly because of a change of its slow component. As expected, the first latency was shortened by
2a coexpression.
3trunc had no effects on fast gating, and
3a took a numerically intermediate position regarding these parameters. The effect of
3trunc on availability and peak current fell visibly short of the effect of
3a in this expression system. Finally, we examined a relevant window of test potentials (0 to +20 mV) where voltage-dependent activation of gating parameters rises steeply. This reveals the biophysical nature of the differential modulation of the pore subunit by
2a versus
3a and
3trunc (Fig. 6). With
2a, the voltage dependence of open probability was shifted to the left. This was not as prominent with
3a and even less with
3trunc. Unfortunately, more positive potentials could not be examined because of bandwidth limitation (29). However, it becomes clear that the amount of voltage shift is larger with
2a. Considering availability, modulation by
2a and
3a again were quite similar (not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to earlier studies that did not detect 3-subunit mRNA expression in human LV mRNA (30), we detected transcripts of 3.5 kb and smaller sizes in RA, RV, and LV from both human non-failing and failing ischemic myocardium. Transcripts of 3.5 kb were also reported for other human tissues (30). Both the expression of
3 transcripts in the whole heart and the finding of differential splicing of exon 6 suggest a physiological role of the
3 in the human heart, which is also supported by our real-time PCR results that revealed a higher
3 copy number when compared with
2a.
Real-time experiments revealed unreckoned prevailing 2b expression when compared with
2a, which suggests an important physiological relevance of the
2b, although it lacks the palmitoylation sequence contained in the
2a. Compared with both
2b and
3,
2a expression level is much lower, which was surprising in view of its attributed important role for modulation of the L-type calcium inward current in the cardiomyocyte (13). However, gene expression levels of the same magnitude were also reported for transcripts of the
1b-subunit isoform as detected by the competitive RT-PCR technique (3). At the moment, the physiological relevance of the different magnitudes in the expression levels of
1b,
2a,
2b, and
3 remains unclear but certainly warrants further investigation.
As with previous gene expression studies in human myocardium (3, 5), expression levels of 2a,
2b, and
3 were all normalized to cardiac calsequestrin expression in the same sample. This sarcoplasmic calcium-binding protein was chosen as "housekeeping gene," because different studies in human heart failure showed that its expression is consistently unchanged at the mRNA and the protein level when compared with non-failing myocardium (21). Furthermore, cardiac calsequestrin is almost exclusively expressed in the cardiomyocyte (31); thus, in human heart expression of the gene of interest can be related to the cardiomyocyte content in the specimen examined.
Cellular localization of the 2b- and
3-subunit isoforms cannot be delineated from our gene expression studies, and appropriate experiments might also be crippled by their low abundance of expression. For this reason, we addressed this question at the functional level by expressing the
3a- and
3trunc-subunit in cells that stably express the CaV1.2a, CaV1.2b, and human CaV1.2. The calcium currents obtained by coexpression with the
3 isoforms were compared with currents induced by rabbit
2a because of its 96% homology to the human
2b. Indeed, the
3a, and to a lesser extent the
3trunc splice variant, elicited functional impact when coexpressed with the cardiac pore-forming subunit. Interestingly, the extent of modulation by
3a was substantially less than with
2a. This difference is not because of a species mismatch between the rabbit and human clones, because similar findings were obtained with both rabbit and human cardiac pore-forming subunits. We also tend to exclude confounding effects of a different dose of the subunit isoforms; all technical precautions (same expression vector, same amount of plasmid DNA) were appropriately implemented, and, more convincingly, the distinctive features of single channel modulation by
2a and
3a help to rule out this concern. Although
2a and
3a equivalently increased channel availability, only
2a showed a large and statistically robust stimulation of the fast gating properties. A more pronounced influence of the same kind of modulation, as with an increased gene dose, should have affected both parameters in parallel. One may argue that our single channel analysis could be biased by presence of a multitude of channels in the patch, even in cases where no multiple simultaneous openings were detected. However, this would even sharpen the distinction between
2a and
3a, because we can exclude multichannel patches with greater certainty in the case of
2a. The probability that the absence of stacked openings indicates the presence of truly one channel is >98% in a typical experiment with
2a but only
92% in case of
3a (32).
The electrophysiological distinction between -subunits is not without precedence. When rat
-subunit-GFP fusion proteins were overexpressed in native myocytes,
2-subunits caused a more prominent increase of whole-cell currents than
3-subunits (33). An abstract (34) and a recent paper (13) showed that
-subunit isoforms can be discriminated regarding closed time distributions at the single channel level, a finding in line with our data.
The observed functional consequence of differential splicing of 3-subunits cannot explain the increase in channel activity as observed in human heart failure (5).
3trunc-subunits, which appear to modulate the pore in a qualitatively similar manner as
3a, have a less pronounced influence in almost every respect. Although channel activity is typically doubled by
3trunc, this effect does not reach significance when corrected for the multiple comparisons we were obliged to perform. Viewed in isolation, even the
3trunc isoform is able to modulate the cardiac pore subunits, albeit in a more subtle manner. This is not unexpected, because N-terminal interaction sequences remain despite the premature translational stop, which results in loss of the principal
-subunit interaction domain and, furthermore, of potential phosphorylation sites for protein kinase C and the casein kinase II (30). Also, such structural features may alter interaction with G-proteins (35, 36).
3trunc expression may actually compete with other isoforms and thus reduce single channel activity in native heteromultimeric complexes, but channels behaving in such a manner were not detected in native failing human myocytes (5, 29, 37).
In summary, -subunit gene products modulate cardiac calcium channel pores in an isoform- and splice variant-specific manner. Therefore, it is crucial to quantify not only the absolute amount of mRNA or protein of these subunits under pathological conditions but rather the relative contribution of the subtypes actually expressed. Future studies should dissect such
-subunit alterations. These could serve as a molecular basis of altered single channel behavior in heart failure, which in turn may critically compromise excitation-contraction coupling.
![]() |
FOOTNOTES |
---|
Contributed equally to this work.
Contributed equally to this work. To whom correspondence may be addressed: Cardiology, Swiss Heart Center Bern, University Hospital, 3010 Bern, Switzerland. Tel.: 41-31-632-8261; Fax: 41-31-632-4560; E-mail: roger.hullin{at}insel.ch. ¶ To whom correspondence may be addressed: Dept. of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Koeln, Germany. Tel.: 49-221-478-6064; Fax: 49-221-478-5022; E-mail: stefan.herzig{at}uni-koeln.de.
1 The abbreviations used are: NF, non-failing; LV, left ventricular; RT, reverse transcriptase; ICM, ischemic cardiomyopathy; RA, right atrium; RV, right ventricle; CHO, Chinese hamster ovary; HEK, human embryonic kidney; GFP, green fluorescent protein; ANOVA, analysis of variance; nn, nucleotide.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|