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(Circulation Research. 1997;81:526-532.)
© 1997 American Heart Association, Inc.

Articles

Alternatively Spliced IS6 Segments of the _1C Gene Determine the Tissue-Specific Dihydropyridine Sensitivity of Cardiac and Vascular Smooth Muscle L-Type Ca²⁺ Channels

Andrea Welling, Andreas Ludwig, Stephanie Zimmer, Norbert Klugbauer, Veit Flockerzi, , Franz Hofmann

From the Institut für Pharmakologie und Toxikologie der Technischen Universität München (Germany) (A.W., A.L., N.K., F.H.), and the Institut für Pharmakologie (S.Z., V.F.), Universität des Saarlandes, Homburg/Saar, Germany.

Correspondence to A. Ludwig, Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Straße 29, 80802 München, Germany. E-mail ludwig{at}ipt.med.tu-muenchen.de

	Abstract

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Abstract Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca²⁺ channel at lower concentrations than the cardiac Ca²⁺ channel, although their ₁ subunit, which binds the DHPs, is derived from the same gene. This _1C gene gives rise to several splice variants, among which the _1C-b variant is affected by lower concentrations of nisoldipine than the _1C-a variant. Functional expression of chimeras of _1C-a and _1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the _1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the _1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 _1C-a and IS6 _1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca²⁺ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the _1C gene.

Key Words: Ca²⁺ antagonist • L-type Ca²⁺ channel • chimeric channel • heart muscle • vascular smooth muscle

	Introduction

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High-voltage–activated Ca²⁺ channels are the pathways for voltage-gated Ca²⁺ entry in heart, smooth muscle, and neurons. These Ca²⁺ channels are multimeric protein complexes composed of up to four different proteins, namely, the ₁, ₂/, ß, and subunits.¹ The ₁ subunit is the principal subunit and contains the voltage-gated channel pore. Six distinct genes have been identified encoding homologous ₁ subunits. The "cardiac" (_1C-a)² and the "smooth muscle" (_1C-b)³ proteins are splice products of the class-C Ca²⁺ channel ₁ gene. These _1C subunits are the major targets for all known Ca²⁺ channel blockers, such as DHPs, phenylalkylamines, and benzothiazepines, which are powerful drugs in the treatment of a wide variety of cardiovascular disorders, including hypertension, angina, and some forms of cardiac arrhythmias. Key to most of these applications is the high degree of specificity of these drugs for the vascular smooth muscle L-type Ca²⁺ channel.⁴

DHPs are the most selective and potent blockers of L-type Ca²⁺ channels. Their affinity for channel block depends on the membrane potential^{5 6} ; ie, the extent of channel block increases with a shift of the membrane potential from -80 mV to more positive values. Myocardial and vascular smooth muscle cells express the same L-type Ca²⁺ channel, which is derived from the _1C gene. The resting potential of most smooth muscle cells is more positive than that of myocardial cells. Therefore, it was suggested that the higher selectivity of DHPs for the vascular smooth muscle channel than for the cardiac channel is caused by the lower resting membrane potential of smooth muscle cells.^{4 7} However, recent reports^{8 9 10} showed that slight differences in the primary sequence of the _1C subunit are sufficient to affect significantly the DHP block of expressed channels. These findings indicated that the well-documented high sensitivity of the smooth muscle Ca²⁺ channel for DHPs might be caused not only by the lower membrane potential of smooth muscle but also by structural differences between the cardiac and vascular L-type Ca²⁺ channel.

The _1C-a and _1C-b channel differ only at four sites constituting 5% of their amino acid sequence, namely, the amino terminus, the transmembrane segments IS6 and IVS3, and an insert in the cytoplasmic loop connecting repeat I and repeat II, which is only present in the _1C-b subunit. Interestingly, photoaffinity labeling studies indicated that the DHPs interact with extracellular sites of the channel close to IS6, IIIS6, and IVS6.^{11 12} Recent studies using site-directed mutagenesis of the _{1C, 1A}, and _1E subunit confirmed the interaction sites at the IIIS5/S6 and IVS6 segment of the _1C subunit.^{13 14 15 16 17 18} In order to define the sequence critical for the distinct DHP sensitivity, all possible chimeras between the _1C-a and _1C-b subunit were constructed and functionally expressed, and their pharmacological properties were studied.

The results demonstrate that the alternatively spliced IS6 segments of the _1C gene are responsible for the different DHP sensitivity of smooth and cardiac muscle L-type Ca²⁺ channels and that the IS6 _1C-a and IS6 _1C-b segments are selectively expressed in cardiac and vascular smooth muscle, respectively.

	Materials and Methods

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Construction of Chimeras
For construction of carboxy-terminal deletion mutants of the _1C-a subunit (termed pcDNA3HK1733) and _1C-b subunit (pcDNA3LK1728), plasmids pcDNA3HK1 and pcDNA3LK1¹⁹ were truncated at amino acids 1733 and 1728, respectively, by a PCR protocol.²⁰ For details of construction, see Seisenberger et al.⁹ To construct chimeras pcDNA3HK2, pcDNA3LK2, pcDNA3LK3, pcDNA3LK4, pcDNA3LK5, pcDNA3LK6, pcDNA3LK7, pcDNA3LK8, pcDNA3LK10, and pcDNA3LK12, the 2510-bp Afl II (nucleotide 2674) of pcDNA3LK1728–Xba I (polylinker site of pcDNA3LK1728) fragment and the following cDNA fragments from pcDNA3HK1733 were used: Afl II (nucleotide 2689)–Xba I (polylinker site of pcDNA3HK1733) fragment, Cla I (nucleotide 257)–Afl II (nucleotide 2689) fragment, Cla I (nucleotide 257)–Dra II (nucleotide 1386) fragment, Dra II (nucleotide 1386)–Nar I (nucleo-tide 1608) fragment, and Mun I (polylinker site of pcDNA3HK1)–Mun I (nucleotide 498) fragment. For the construction of pcDNA3LK7 and pcDNA3LK8, the EcoRI (nucleotide -11)–EcoRI (nucleotide 2199) fragment of pcDNA3LK1 was ligated into pBluescript II (Stratagene) to obtain pBlueLK1EcoRI. The Cla I–Dra II fragment was removed and replaced by the corresponding fragment of pcDNA3HK1, yielding pcDNA3LK7. To obtain pcDNA3LK8, the Dra II–Nar I fragment of pcDNA3HK1 was inserted into pBlueLK1EcoRI, and the resulting fragment was reinserted in pcDNA3LK1728.

Transfection of HEK 293 Cells
HEK 293 cells were transiently transfected with the expression vectors for the various chimeras (0.5 µg) by a lipofection method using lipofectamine according to the manufacturer (GIBCO-BRL Life Technologies). After transfection, the cells were grown for 16 to 18 hours, washed with PBS, and grown for another 24 hours before beginning the electrophysiological experiments.

Electrophysiological Recordings
I_Ba was measured at room temperature using the whole-cell patch-clamp technique. The external solution contained (mmol/L) NaCl 82, TEA-Cl 20, BaCl₂ 30, CsCl 5, MgCl₂ 1, EGTA 0.1, glucose 10, and HEPES 5, pH 7.4 (NaOH). The pipette solution contained (mmol/L) CsCl 102, TEA-Cl 10, EGTA 10, MgCl₂ 1, ATP 3, and HEPES 5, pH 7.4. The investigated cell was superfused constantly by the external solution. The fast capacitive component was compensated after forming the seal; the slow component and the series resistance were compensated after breaking into the whole-cell configuration. The leak current was compensated by subtracting the adequate multiple of a linear component obtained by small depolarizing voltage steps from a negative HP. Data were sampled at 5 kHz and filtered at 1 to 3 kHz. Current inhibition by nisoldipine was measured during trains of 40-ms test pulses applied once every 5 s as described previously.^{6 8 9} Rested-state channel block (protocol A) was measured by applying 40-ms test pulses once every 5 or 30 s from -80 mV to +20 or +30 mV. The developed block is termed frequency-independent block. In protocol B, channel block was measured after changing the HP from -80 to -40 mV (onset of inactivated-state channel block), and recovery from inhibition was studied by returning the HP to -80 mV (recovery from inactivated-state channel block).^{6 8} The test pulse voltage was either +20 or +30 mV, according to the peak of the current-voltage relation under these experimental conditions. Usually, inactivated- and rested-state channel blocks were each measured in different experiments with a single drug concentration. Nisoldipine was diluted to the final concentrations from a stock solution of 20 mmol/L in ethanol into the external solution and applied with a rapid solution exchanger. Data plotting and statistical analysis was carried out using ORIGIN software (Microcal). Pooled data are given as mean±SEM.

RT-PCR and Northern Blot Analysis
Total RNA from rat heart and aorta was isolated by the guanidinium thiocyanate method, and poly(A⁺) RNA was separated by oligo(dT) cellulose chromatography (Stratagene). Poly(A⁺) RNA was reverse-transcribed using oligo(dT) primers. PCR amplification for 40 cycles was done with a primer pair flanking the IS6 segment (primer 1 [nucleotides 1343 to 1362] and primer 2 [nucleotides 1477 to 1497]) of _1C-a.²¹ Reaction products were characterized by restriction mapping with several enzymes, including BamHI and Hae III, cloned in a pUC19-derived vector, and sequenced on both strands. For Northern blotting, 10 µg poly(A⁺) RNA from rat heart and aorta was electrophoresed on a 1.2% agarose gel, and equal RNA loading was confirmed by ethidium bromide staining of the gel. Separated RNA was transferred to Hybond-N nylon membranes (Amersham) and hybridized under high stringency with random-primed labeled cDNA probes. The probes were the 154-bp fragments corresponding to the type-a and type-b IS6 segments of the rat _1C sequence. The blot was exposed to BAS-III image plates (Fuji) and Hyperfilm MP (Amersham). Signals on the image plate were quantified with a BAS-1500 phosphorimager (Fuji). For rehybridizations, the blot was stripped in 0.1x SSC and 0.1% SDS at 95°C for 30 minutes, and complete removal of the probe was checked by autoradiography.

In Situ Hybridization
Adult Sprague-Dawley rats (250 to 350 g) were anesthetized with sodium pentobarbital (60 mg/kg IP). The heart was removed, frozen in isopentane, and cut at 12-µm thickness in a cryostat. Sections were fixed with 4% paraformaldehyde in PBS, pH 7.4. Slices were pretreated with proteinase K, acetylated with 0.25% acetic anhydride, prehybridized for 2 hours at 42°C in hybridization buffer (10 mmol/L Tris [pH 8.0], 0.3 mol/L NaCl, 1 mmol/L EDTA, 1x Denhardt's solution, 10% dextran, 50% deionized formamide, and 50 mmol/L dithiothreitol), and hybridized with ³⁵S-UTP–labeled cRNA probes (5x10⁶ cpm/mL hybridization buffer) for 16 hours at 55°C. Slides were washed in 2x SSC, incubated in RNase A, and washed then in 0.1x SSC at 70°C. Sections were dipped in autoradiography emulsion Kodak NTB-2, exposed for 6 weeks, counterstained with hematoxylin/eosin, and examined with dark-field illumination. cRNA probes were in vitro–transcribed from the two IS6 segment variants cloned in a pUC 19–derived vector. The _{1C common} probe is directed against nucleotides 2745 to 3022 in the loop region between IIS6 and IIIS1 of the _1C-a subunit.²¹

	Results

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Electrophysiological Characterization of the Ca²⁺ Channel Chimeras
A previous analysis of the two splice variants of the _1C gene, the cardiac _1C-a and the smooth muscle _1C-b protein, showed that low concentrations of nisoldipine induced a rested-state channel block only in the smooth muscle _1C-b channel; conversely, recovery from inactivated-state block was significantly greater in the _1C-a than the _1C-b channel.⁸ The native cardiac L-type Ca²⁺ channel had the same properties as the expressed _1C-a channel, suggesting that the different DHP sensitivity of the cardiac and smooth muscle L-type Ca²⁺ channel may reside in structural differences. The two splice variants of the _1C gene differ at the four regions, A, B, C, and D (Fig 1A). To identify the region responsible for the different nisoldipine sensitivity, all possible chimeras of _1C-a (named HK1) and _1C-b (named LK1) subunits were constructed (termed LK2 to LK8, LK10, LK12, and HK2) (Fig 1B) and functionally expressed in the absence of auxiliary subunits. All channels were carboxyterminal-truncated, since the truncated _1C subunit yields higher I_Ba without affecting the basic channel kinetics.^{9 20 22}

View larger version (20K): [in this window] [in a new window]   Figure 1. Schema of the chimeric Ca2+ channel constructs derived from the 1C-a (HK1) and 1C-b (LK1) sequence. A, Proposed structure of the 1C subunit of the L-type Ca2+ channel. I, II, III, and IV indicate suggested repeats of the Ca2+ channel; +, amphipathic helix and voltage sensor; and A, B, C, and D, differences in the sequence of the 1C-a and 1C-b splice variant. B, Schematic figures of chimeric constructs between HK1 and LK1. The black boxes represent the sequences from the 1C-b subunit. All constructs were carboxyterminal-truncated as shown in panel A.

Initial electrophysiological characterization of these chimeras indicated that indeed the truncated _1C chimeras induced regular L-type I_Ba. Neither the current-voltage relations nor the steady state inactivation curves differed significantly from those obtained previously with the full-length or truncated _1C-b or _1C-a subunit.^{8 9 22} I_Ba started at membrane potentials positive to -30 mV, was maximal between +20 and +30 mV, and reversed at +75 mV. Half-maximal inactivation under steady state conditions occurred between -8 and -9 mV. The maximal current densities of the truncated channels LK1 and HK1 were 18±1.2 pA/pF (n=22) and 8±0.7 pA/pF (n=20), respectively (Fig 2). Comparison of the current densities obtained with the different chimeras showed that LK2 (19 pA/pF), LK4 (25 pA/pF), LK8 (13 pA/pF), and LK12 (19 pA/pF) had current densities similar to LK1 (Fig 2). LK5 (5 pA/pF), LK6 (7 pA/pF), HK2 (8 pA/pF), and HK1 (8 pA/pF) had significantly smaller current densities, and LK3 (30 pA/pF), LK7 (39 pA/pF), and LK10 (19 pA/pF) had significantly higher current densities than LK1. All constructs with the "cardiac" amino terminus (region A) had low I_Ba densities, whereas intermediate current densities correlated with the "lung" amino terminus. The highest current densities were obtained with the combination of the lung amino terminus (region A) and the cardiac IS6 segment (region B) (Fig 2). Obviously, not only the carboxy terminus^{20 22} but also the amino terminus of the _1C proteins influences the current density of the expressed _1C channels.

View larger version (16K): [in this window] [in a new window]   Figure 2. Current densities obtained by the genuine and chimeric channels. Values are given as mean±SEM, with the number of experiments shown above the bars. °°P<.01 and °°°P<.001 indicate significantly larger than LK1; **P<.01 and ***P<.001 indicate significantly smaller than LK1.

Nisoldipine Sensitivity of the Chimeras
The sensitivity of the chimeras against the nisoldipine block was tested by the two protocols, which were used and tested in previous studies,^{8 9} to distinguish between smooth muscle and cardiac type of block. In protocol A, the rested-state channel block (protocol designed to measure also frequency-independent block) was tested by superfusion of cells expressing genuine or chimeric channels with 1 or 10 nmol/L nisoldipine at the resting membrane potential of -80 mV and a stimulation frequency of 0.2 (not shown) or 0.033 Hz (see Figs 3 and 4). Nisoldipine reduced I_Ba of the genuine LK1 channel at a concentration of only 1 nmol/L and an HP of -80 mV, even at the very low stimulation frequency of 0.033 Hz (Fig 3). The current was blocked to the same amount at 0.2 Hz (not shown). Such a rested-state channel block was not observed with the cardiac HK1 channel (Fig 4). Further analysis showed that 1 nmol/L nisoldipine exerted a rested-state channel block in chimeras LK2, LK5, LK6, LK8, and LK12, whereas I_Ba of LK3, LK4, LK7, LK10, and HK2 was hardly or not blocked at all (Fig 5). Close inspection of the chimeras revealed that a rested-state channel block occurred in parallel with the presence of the IS6 segment of the _1C-b sequence (segment B in Fig 1A ) but not with any of the other three alternatively spliced regions. In protocol B, the presence and extent of the inactivated-state channel block and the recovery from this block were tested. As already shown in previous studies,^{8 9} a shift of the HP from -80 to -40 mV decreased I_Ba by 20% in both channels in the absence of nisoldipine. In agreement with these previous results, 1 nmol/L nisoldipine reduced I_Ba to 55% and 67% for the cardiac versus the smooth muscle channel, respectively, and 10 nmol/L nisoldipine reduced I_Ba close to zero in both channels (Figs 3 and 4). The time course of the onset of block was faster in the LK1 than in the HK1 cells in the presence of 1 or 10 nmol/L nisoldipine (compare Figs 3 and 4 for 10 nmol/L drug), but the differences for both onset or extent of block at -40 mV in the presence of 1 or 10 nmol/L nisoldipine were not statistically significant. In agreement with these electrophysiological results, the DHP isradipine bound in radioligand studies to both channels with the same K_d value (0.1 nmol/L).²³

View larger version (20K): [in this window] [in a new window]   Figure 3. Nisoldipine (NIS) block of the 1C-b Ca2+ channel (LK1). Top, Time course of IBa (cell capacity, 27 pF; 21 pA/pF) elicited from an HP of -80 mV (filled symbols) or -40 mV (open circles). The times of NIS application are indicated by the black line. Pulse length was 20 ms between b-c and d-e and 40 ms during the remaining experiment. The pulse length was shortened to 20 ms to prevent inactivation during depolarization. The depolarization frequency was decreased to 0.033 Hz between a and b to prevent use-dependent block. Bottom, Individual traces taken at the time points indicated in the top panel.

View larger version (19K): [in this window] [in a new window]   Figure 4. Nisoldipine (NIS) block of the 1C-a Ca2+ channel (HK1). Top, Time course of IBa (cell capacity, 41 pF; 8.5 pA/pF) elicited from an HP of -80 mV (filled symbols) or -40 mV (open circles). Bottom, Individual traces taken at the time points indicated in the top panel.

View larger version (16K): [in this window] [in a new window]   Figure 5. Extent of frequency-independent block. The extent of frequency-independent block was determined at the end of the nisoldipine superfusion (3 minutes) and represents the part of the current blocked in the presence of nisoldipine at an HP of -80 mV, a pulse length of 20 ms, and a frequency of 0.033 Hz divided by the maximal current in the absence of nisoldipine at a frequency of 0.2 Hz and an HP of -80 mV. The extent of block is given as mean±SEM, with the number of experiments shown above the bars.

However, the _1C-a and the _1C-b channels and the chimeras differed significantly in their recovery from the inactivated-state channel block. Therefore, this criterion was used in further experiments to distinguish the different types of inhibition. In the presence of 1 nmol/L nisoldipine, the reversal of the membrane potential from -40 to -80 mV unblocked almost 70% of the current in the HK1-expressing cells but only 47% in the LK1-expressing cells. The reversal of the membrane potential in the presence of 10 nmol/L nisoldipine revealed the clearest difference with an unblock of almost 50% in the HK1-expressing cells but only a minimal amount of current in the LK1-expressing cells (Fig 6). After reversal of the membrane potential from -40 to -80 mV in the presence of 10 nmol/L nisoldipine, only 15% to 22% of I_Ba was recovered in cells expressing LK1 and the chimeras LK2, LK5, LK6, LK8, and LK12, whereas 41% to 46% of I_Ba was recovered in cells expressing HK1 and the chimeras LK4, LK7, and HK2. The extent of recovery of the chimeric channel LK10 was with 32% less than that of the HK1 channel, but this value was still significantly higher than that of the LK1 channel. Inspection of all chimeric channels indicated again that chimeras that contained the IS6 segment of the _1C-b subunit had a poor recovery from voltage-dependent block, whereas those that contained the IS6 segment of the _1C-a subunit had a good recovery. The only exception was chimera LK3. The current of this channel recovered by 27%, which is a higher recovery than that of the LK1 channel but lower than that of the HK1 channel. Together, these results suggested that all chimeras retained the property of a voltage-dependent DHP block. However, similar to the in vivo situation,⁸ the cardiac _1C-a channel recovered better from the voltage-dependent nisoldipine block than did the smooth muscle _1C-b channel. This difference was caused by the primary sequence of the IS6 segment.

View larger version (17K): [in this window] [in a new window]   Figure 6. Recovery from block. The recovery from block represents the part of the current that redeveloped after returning the holding potential from -40 to -80 mV in the presence of nisoldipine divided by the current before shifting the HP from -80 to -40 mV (see Figs 3 and 4). The values are mean±SEM, with the number of experiments shown above the bars. **P<.005 and ***P<.001 indicate significantly different from LK1.

Tissue-Specific Expression of the IS6 Segments
The above results indicated that the use of alternative IS6 segments significantly affected the DHP sensitivity of the _1C subunit. It was of great importance to see whether the two IS6 segments were expressed selectively in cardiac and smooth muscle. To address this question, the 154-bp fragment encoding transmembrane segment IS6 was RT-PCR–amplified from rat heart and aorta poly(A⁺) RNA (Fig 7A). The sequence of the amplified fragment from rat heart was 96.8% and 100% identical at the nucleotide level to the corresponding sequences cloned from rabbit heart² and rat brain,²¹ respectively, demonstrating that it encodes the type-a variant of the IS6 segment. In contrast, the fragment amplified from rat aorta showed 98.7% and 99.4% identity at the nucleotide level to the type-b variant of IS6 originally cloned from rabbit lung³ and mouse brain,²⁴ respectively. The PCR products from each tissue were digested with the restriction enzymes BamHI and Hae III. BamHI cuts only the IS6 segment of the _1C-a sequence, and Hae III cuts only the IS6 segment of the _1C-b sequence. This analysis demonstrated almost tissue-specific representation of the IS6 segment in heart and aorta RNA (Fig 7A, lanes 1 to 4). To further characterize the relative abundance of the two IS6 variants, the amplicons were subcloned and analyzed by restriction digestion. Eighteen of 20 cardiac PCR clones contained the IS6 segment of the _1C-a sequence, and only 2 contained the IS6 segment of the smooth muscle _1C-b sequence. In contrast, 17 of 20 aorta PCR clones had the sequence of the smooth muscle type-b IS6 segment. These results supported the hypothesis that both IS6 splice variants were expressed in a tissue-specific manner, with IS6 _1C-a being present in heart and IS6 _1C-b being present in smooth muscle cells.

View larger version (61K): [in this window] [in a new window]   Figure 7. Tissue-specific expression of the two IS6 segments. A, RT-PCR analysis. RT-PCRs with rat aorta (A) or heart (H) RNA as template were analyzed by digestion with Hae III (Hae) or BamHI (Bam). B, Northern blot analysis. Poly(A+) RNA (5 µg) from rat heart and poly(A+) RNA (10 µg) from rat aorta were electrophoresed. The blot was hybridized with a 1:1 mixture of the two IS6 segment–specific probes (a), the probe for the IS6 segment of the 1C-a subunit (b), and the probe directed against the IS6 segment of the 1C-b subunit (c). C, In situ hybridization of rat heart sections, dark-field views. Sections were labeled with the 1C common antisense probe (a), the 1C common sense probe (b), the riboprobe directed against the IS6 segment of the 1C-a subunit (c), and the riboprobe specific for the IS6 segment of the 1C-b subunit (d). The arrow marks a coronary artery. Bar=200 µm for panel C, a to d.

To provide an independent confirmation of the expression level of the two IS6 splice variants, Northern blot analysis of rat heart and aorta poly(A⁺) RNA was performed (Fig 7B). A 1:1 mixture of the two IS6 segment–specific probes detecting the entire pool of IS6 segment splice variants hybridized with transcripts of 8.5 and 14 kb in both tissues (Fig 7B, blot a). Obviously, heart poly(A⁺) RNA contained a higher proportion of _1C mRNA than did aorta poly(A⁺). The blot was stripped and rehybridized with the probe recognizing the IS6 segment of the _1C-a subunit. Transcripts of 8.5 and 14 kb were labeled exclusively in cardiac poly(A⁺) RNA (Fig 7B, blot b). In contrast, rehybridizing with a probe specific for the IS6 segment of _1C-b detected transcripts of 8.5 and 14 kb in aorta poly(A⁺)RNA and, to a smaller extent, also in cardiac poly(A⁺) RNA tissues (Fig 7B, blot c). These signals were quantified using a phosphorimager. Based on the finding that nearly 100% of IS6 segments detected in aorta poly(A⁺) RNA were of the smooth muscle type (Fig 7B, blot b), comparison of the relative band intensities of blots a and c showed that heart poly(A⁺) RNA contained 79% of the cardiac-type and 21% of the smooth muscle–type IS6 segments.

In Situ Hybridization
Finally, the tissue-specific expression of the two _1C splice variants was tested by in situ hybridization on rat heart sections. Hybridization of a frontal section through the heart ventricles with an _{1C common} probe detecting both type a and type b of the _1C gene showed strongly scattered labeling of ventricular myocytes and coronary arteries (Fig 7C, a). As a control, labeling of an adjacent section with the corresponding sense riboprobe showed no signal (Fig 7C, b). A cRNA probe directed against IS6 _1C-a strongly labeled ventricular myocytes but yielded no signal over different coronary arteries (Fig 7C, c). In contrast, the probe specific for the IS6 segment of the _1C-b subunit strongly labeled the wall of the coronary arteries, whereas only weak labeling of ventricular myocytes was detected (Fig 7C, d). Similar results as described above were seen with in situ hybridization of the three probes on sections through heart atria. Only IS6 _1C-b was detected in sections through the aorta (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of this and two previous studies^{8 9} showed that the nisoldipine sensitivity of the a and b splice variants of the _1C gene is different. This difference has been observed (1) with the full-length _1C-a and _1C-b subunits stably expressed in Chinese hamster ovary cells,⁸ (2) with the carboxyterminal-truncated _1C-a and _1C-b subunits stably expressed in HEK 293 cells,⁹ and (3) with the transiently expressed, carboxyterminal-truncated _1C-a and _1C-b subunits (the present study). The different nisoldipine sensitivity of the two splice variants is due to the presence of different IS6 segments within the _1C-a and _1C-b proteins. PCR amplification, Northern analysis, and in situ hybridization showed that the a and b splice variants of the IS6 segment are expressed in a tissue-specific manner. The IS6 segment of the _1C-a subunit is exclusively expressed in cardiac myocytes, whereas the IS6 segment of the _1C-b subunit is expressed in vascular smooth muscle and, to a small extent, also in cardiac myocytes. This tissue-specific expression of the isoforms of the IS6 segment clearly demonstrates that the preferential action of DHPs on the vascular L-type Ca²⁺ channel is caused at least partially by the structural properties of the _1C subunit present in vascular smooth muscle cells.

The major difference between the a_1C-a and a_1C-b channels was that (1) low concentrations of nisoldipine alone caused an frequency-independent block of the _1C-b channel, and (2) recovery from the inactivated-state channel block was observed readily with only the cardiac _1C-a, but not the smooth muscle _1C-b, channel. These findings suggested that nisoldipine interacted not only with the inactivated but also with the resting or closed state of the _1C-b channel. Moreover, the occurrence of a rested-state channel block indicates that the DHP block of the _1C-b channel has a voltage-independent component that is not apparent with the cardiac _1C-a channel. The association of the rested-state channel block with the presence of the IS6b segment that is part of the smooth muscle Ca²⁺ channel is in excellent agreement with the physiology of this tissue. A change in vascular smooth muscle tone is obtained without large changes in the membrane potential.⁷ The smooth muscle membrane potential remains relatively constant for long time periods. Under such conditions, a rested-state channel block will be more efficient than a block depending on strong depolarization of the membrane potential. In contrast to the vascular smooth muscle channel, the cardiac channel containing the IS6a segment showed no rested-state channel block and an excellent recovery from inactivated-state channel block. This property is in agreement with the physiology and pharmacology of the native cardiac L-type Ca²⁺ channel showing a frequency-dependent increase in the DHP block. The increase in frequency shifts the equilibrium between the different channel states to the inactivated state.

In general, the results of the present study confirm previous findings that the DHP sensitivity of both channels is different. As observed also by others,¹⁰ the difference between both channels is less apparent at depolarized membrane potentials but significant at more hyperpolarized membrane potentials. In agreement with these functional results, the DHP isradipine binds to both channels with the same K_d value (0.1 nmol/L).²³ In these binding studies, the channel is believed to be in its inactivated state.^{5 6} The amino acid residues that are required for the high-affinity interaction with DHPs have been identified in the IIIS5, IIIS6, and IVS6 segments.^{13 14 15 16 17 18} Both channels have the same sequence in these regions, explaining the identity of the two binding constants and the similarity in the extent of the nisoldipine block at a HP of -40 mV.

The IS6 segment has been implicated in the process of voltage-dependent inactivation of the channel.^{25 26} Furthermore, it has been reported¹² that amino acid residues close to IS6 of the _1S subunit interact with DHPs. These previous findings lend further support to the hypothesis that the IS6 segment is involved in a "low-affinity" interaction with nisoldipine and other DHPs that results in a rested-state block of the channel. The rested-state channel block may require interaction with critical amino acid residues of the IIIS5, IIIS6, and IVS6 region of the _1C subunit. This low-affinity interaction is modulated by the use of the alternative IS6 segments. The results of the present study suggest that the high sensitivity of the smooth muscle Ca²⁺ channel for DHPs is caused not only by the more depolarized potentials of vascular smooth muscle but additionally by the splice variant type b of the IS6 segment, which is specifically expressed in this tissue.

	Selected Abbreviations and Acronyms

 

DHP = dihydropyridine HEK = human embryonic kidney HP = holding potential IBa = Ba2+ current PCR = polymerase chain reaction RT = reverse transcription TEA = tetraethylammonium

	Acknowledgments

 
This study was supported by SET (Stiftung zur Förderung der Erforschung von Ersatz- und Ergänzungsmethoden zur Einschränkung von Tierversuchen), Deutsche Forschungsgemeinschaft, and Fond der Chemie. We thank Barbara Lehnert and Sabine Stief for excellent technical assistance and Eva Roller for the graphical work.

	Footnotes

 
© 1997 American Heart Association, Inc.

Received April 14, 1997; accepted July 22, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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