Journal of Histochemistry and Cytochemistry, Vol. 48, 807-820, June 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Immunodetection of {alpha}1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

Marco Weiergräber1,a, Alexey Pereverzev1,a, Rolf Vajnaa, Margit Henrya, Martin Schramma, Wolfgang Nastainczykb, Heike Grabschc, and Toni Schneidera
a Institute of Neurophysiology, University of Köln, Köln, Germany
b Institute of Medical Biochemistry and Molecular Biology, University of Saarland, Homburg/Saar, Germany
c Institute of Pathology, Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany

Correspondence to: Toni Schneider, University of Köln, Institute of Neurophysiology, Robert-Koch-Str. 39, D-50931 Köln, Germany. E-mail: Toni.Schneider@uni-koeln.de


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The calcium channel {alpha}1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of {alpha}1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac {alpha}1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to {alpha}1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of {alpha}1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of {alpha}1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of {alpha}1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of {alpha}1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin. (J Histochem Cytochem 48:807–819, 2000)

Key Words: {alpha}1E calcium channels, R-type Ca2+ channel, endocrine, chromogranin, atrial natriuretic peptide, urodilatin, H9c2 cells, conducting system, blood pressure regulation


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cardiomyocytes express T- and L-type voltage-gated Ca2+ channels (Nilius 1998 ). T-type channels support the electrogenesis of repetitive activity, whereas L-type channels and, to some extent, also T-type channels (Sipido et al. 1998 ), participate directly in Ca2+-induced Ca2+ release during contraction of the heart (Nilius 1998 ). Cardiac L-type calcium channels are well characterized on the functional (Nilius et al. 1985 ; Trautwein et al. 1986 ; Bean 1989 ; Hess 1990 ; Vassort and Alvarez 1994 ), biochemical (Ferry et al. 1987 ; Schneider and Hofmann 1988 ; Haase et al. 1991 ), and molecular levels (Mikami et al. 1989 ; Wyatt et al. 1997 ). Disruption of the cardiac {alpha}1C-containing L-type Ca2+ channel by gene targeting is lethal before birth (Seisenberger et al. 1999 ). In addition to {alpha}1C, {alpha}1D has also been detected by antibodies in functional studies of guinea pig heart (Wyatt et al. 1997 ).

Using electrophysiological methods, cardiac T-type Ca2+ channels were detected not only in sinoatrial node cells (Irisawa et al. 1993 ) but also in atrial (Bean 1985 ) and ventricular cells from different species (Nilius et al. 1985 ; Tytgat et al. 1988 ; Sipido et al. 1998 ). In rat ventricular cells, T-type channels are not as easily identified as they are in guinea pig. Recently, however, T-type Ca2+ channels were recorded on the single-channel level in rat ventricular myocytes (Handrock et al. 1999 ). This functional difference between closely related species (Bers and Perez-Reyes 1999 ) is rather unexpected and is not yet well understood (Nilius 1998 ).

Three new T-type voltage-gated Ca2+ channels ({alpha}1G, {alpha}1H, and {alpha}1I) have recently been cloned and expressed in recombinant systems (Cribbs et al. 1998 ; Perez-Reyes et al. 1998 ; Lee et al. 1999 ; Mittman et al. 1999 ). Some of their basic electrophysiological properties are similar to those of known myocardial T-type calcium channels, such as their small, tiny conductance, and the transient time course.

The detection by Northern blotting of two of the three cloned low-voltage-activated Ca2+ channel subunits ({alpha}1G, {alpha}1H) in heart (Cribbs et al. 1998 ; Perez-Reyes et al. 1998 ) has increased the number of putative candidates for cardiac T-type Ca2+ channels. Originally, the IGF-induced T-type Ca2+ current in heart has been related to {alpha}1E-induced Ca2+ inward currents, because antisense oligonucleotides against {alpha}1E mRNA inhibited the IGF-induced T-type current (Piedras-Renteria et al. 1997 ). This T-type current is believed to be important mainly during development (Kawano and DeHaan 1991 ; Xu and Best 1992 ). Because recombinant {alpha}1E is not considered a typical T-type current (Randall and Tsien 1997 ; Nakashima et al. 1998 ), the immunodetection of {alpha}1E as a method independent of RT-PCR amplification (Piedras-Renteria et al. 1997 ; Mitchell et al. 1999 ) should give additional insight into a possible physiological role for {alpha}1E in heart. Therefore, the distribution of {alpha}1E was compared with the expression of neuroendocrine markers (chromogranin A/B) in the myocardium. Furthermore, the distribution of {alpha}1E was investigated in human kidney because this is another organ that contains natriuretic peptides and plays an important role in blood pressure regulation.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

RNA isolation and RT-PCR
Total RNA was isolated from fresh or from shock-frozen rat brain (cerebrum and cerebellum) or from rat heart using standard protocols (Chomczynski and Sacchi 1987 ).

Fragments of cDNA were amplified by PCR (Perkin–Elmer Cetus Instruments; Norwalk, CT) after reverse transcription of total RNA using standard protocols as specified (Schneider et al. 1994 ; Pereverzev et al. 1998 ). The sequences of primer pairs and the annealing temperatures used for the PCR reactions of {alpha}1 subunits from high-voltage-activated Ca2+ channels and for the housekeeping enzyme hypoxanthine phosphoribosyltransferase have been summarized recently (Vajna et al. 1998 ). The rat {alpha}1G cDNA fragment was amplified using the primer Gfrwsp, 5'-AGCCCCGGTGGTTTCTTCTA-3' (nt 650–670) of rat {alpha}1G cDNA sequence (GenBank AF027984; Bethesda, MD) as forward and Grevsp, 5'-TGAGCGGTCGCAGCACAC-3' as reverse primer (nt 1047–1030). The rat {alpha}1H cDNA fragment was amplified using oligo-452341, 5'-AGGAGGCTC-GGCGCCGG-3' (homologue to human {alpha}1H, AF051946, nt 4777–4793) as forward and oligo-452340, 5'-GGATAGGAGGACGATGGCCAA-3' (homologue to human {alpha}1H, nt 5131–5150) as reverse primer. The rat {alpha}1I cDNA fragment was amplified using Ifrwis, 5'-GCTGCGGCGCCTGGAAAAGAA-3' (nt 4501–4521) of rat {alpha}1I cDNA sequence (GenBank AF086827) as forward and Irevis, 5'-GCCCATGCACGGACAGCAGCACAAT-3' as reverse primer (nt 4838–4814). The annealing temperatures were 57C for {alpha}1G and {alpha}1H, and 60C for {alpha}1I cDNA fragments.

Oligonucleotide primers were purchased from Eurogentec Bel SA (Seraing, Belgium) or from Gibco/Life Science (OligoGold; Gaithersburg, MD).

Subcloning and Sequencing
Amplified cDNA fragments of critical regions were subcloned into the pCR2.1 TOPO vector according to the recommendations of the manufacturers and including the TOPO TA cloning kit (Invitrogen; Leek, The Netherlands). Plasmid DNA was sequenced according to the method of Sanger et al. 1977 on an ABI Prism 377 DNA Sequencer (Perkin Elmer/Applied Biosystems) using the Taq FS dye deoxyterminator cycle sequencing method.

Antibody Production
Two peptides homologous to human {alpha}1E sequences were coupled to hemocyanin and used for immunization of rabbits. Peptide Nast-195 corresponds to a common sequence in all published {alpha}1E isoforms and peptide Nast-197 is part of a 43-amino-acid extension in the carboxy terminus of the fetal brain-derived {alpha}1Ed isoform (Schneider et al. 1994 ; Pereverzev et al. 1998 ), which is also located in two other deduced longer {alpha}1E isoforms, {alpha}1Ee and {alpha}1Ef (Vajna et al. 1998 ). The peptides (a) Nast-195, SGILEGFDPPHPCGVQGC (aa 256–273, in the loop IS5 to pore region; GenBank L27745), and (b) Nast-197 GIYLPSDTQEHAG[C] (aa 1981– 1993, in the carboxy terminus) were synthesized by the solid-phase method (Merrifield 1986 ). For the peptide Nast-197, both the rat and the mouse sequence show one amino acid difference, with a proline instead of a serine residue, compared to the human {alpha}1E sequence. Peptide Nast-197 contains one additional cysteine residue for coupling to a matrix, as shown in brackets. Immunization of the rabbits with the hemocyanin-coupled peptides was described previously (Pereverzev et al. 1998 ; Grabsch et al. 1999 ).

The sensitivity and specificity of the anti-{alpha}1E sera were tested by immunoblots of microsomal membrane proteins from stably transfected HEK-293 cells, which express the human {alpha}1Ed (cell line HEK-2C6 or HEK-2C2), or the deletion mutant {alpha}1Ed-CDEL (cell line HEK-{alpha}1E-CDEL; see Mehrke et al. 1997 ).

Preparation of Paraffin-embedded Tissue and Immunostaining of Tissues and H9c2 Cells
The procedures used in the present study that involved tissues from animals and human were performed in accordance with the regulations governing their use in scientific research.

Female Wistar rats weighing 180–250 g were anesthetized with CO2, decapitated, and the organs rapidly removed. Tissue sections from human kidney were obtained from surgically resected specimens. All tissues were fixed in 10% PBS–buffered formalin at room temperature overnight and embedded in paraffin (Grabsch et al. 1999 ). Immunohistochemistry was performed in 4 µm deparaffinized serial tissue sections.

Before immunostaining, sections underwent a microwave-based heat-induced epitope retrieval (HIER) treatment in a solution of 10 mM citrate buffer, pH 6.0. Endogenous peroxidase activity was quenched by putting the slides in 0.3% hydrogen peroxide in methanol for 20 min at 37C. After washing with PBS, sections were immunostained by the streptavidin–biotin–horseradish peroxidase technique (Grabsch et al. 1999 ) according to the instructions of the manufacturer (Super Sensitive detection system from BioGenex; Hamburg, Germany). The polyclonal antibodies were used at a dilution of 1:100–1:300 in heart, 1:300 in H9c2 cells, and 1:20 in kidney. 3,3'-diaminobenzidine (DAB) was used as chromogen with and without copper sulfate enhancement. The polyclonal antibodies against rat chromogranin A and B were purchased from Quartett (Berlin, Germany). Two negative control procedures were performed. Either the primary antibodies were replaced by preimmune serum or the serum was preincubated with an excess of the peptide (20 µM) used for immunization. During preincubation, we also included in separate experiments hemocyanin (20 µM), which was used as a carrier of peptides during immunization, and myosin (20 µM), an abundant protein in heart, with no effect on the positive staining observed with anti-{alpha}1E.

In some experiments, the purified anti-{alpha}1E serum against an {alpha}1E fusion protein (Volsen et al. 1995 ) and a monoclonal antibody against {alpha}1E, a gift from Dr. R.E. Beattie and Dr. S.G. Volsen (E. Lilly; Windlesham, UK), were used as additional antibodies to compare the staining results with different primary antibodies.

Isolation of Membrane Proteins and Western Blot Analysis
Stably transfected HEK-293 cells were grown as described (Pereverzev et al. 1998 ). Microsomal membrane proteins from untransfected and stably transfected HEK-293 cells and from the rat insulinoma cell line INS-1 (Asfari et al. 1992 ) were isolated according to Pereverzev et al. 1998 . The rat myoblast cell line H9c2 was grown in DMEM supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Microsomal membrane proteins from rat and guinea pig heart and from rat cerebellum and rat kidney were isolated as described previously (Flockerzi et al. 1986 ; Schneider et al. 1992 ). Aliquots of microsomal membranes were stored at -80C.

The membrane proteins were separated by polyacrylamide gel electrophoresis according to standard protocols and immunoblotting was performed as reported (Pereverzev et al. 1998 ). The ECL detection kit was purchased from Amersham (Braunschweig, Germany). Crossreactivity of the anti-{alpha}1E-spec serum was not observed with the shorter splice variant {alpha}1E-CDEL (Nakashima et al. 1998 ; Pereverzev et al. 1998 ) nor with {alpha}1G-, {alpha}1H-, or {alpha}1I-transfected cell lines.

Protein concentrations were determined with the BCA method (Pierce; Rockford, IL). The specificity of the sera was also tested by immunostaining untransfected and stably transfected HEK-293 cells expressing the cloned human {alpha}1E (Schneider et al. 1994 ).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Transcripts of Voltage-gated Ca2+ Channels in Rat Heart
Total RNA was extracted from freshly isolated rat heart and reverse transcribed. Oligonucleotide primer pairs were used to amplify eight different {alpha}1 subunits either from rat cerebrum as a reference or from rat heart cDNA (Fig 1). The transcripts of two L-type {alpha}1 subunits, {alpha}1C and {alpha}1D, were detected. In addition, fragments of two non-L-type ({alpha}1A and 1E) and two T-type subunits ({alpha}1G and {alpha}1H) were amplified (Fig 1). In the cardiac rat cell line H9c2, neither {alpha}1D, {alpha}1A, nor {alpha}1H was observed, but low amounts of {alpha}1I were detected (Fig 1). For {alpha}1C and {alpha}1H, additional splice variants were deduced by the size of the amplified fragment. No transcripts were amplified with primer pairs for {alpha}1S in adult rat heart (not shown).



View larger version (56K):
[in this window]
[in a new window]
 
Figure 1. Detection of {alpha}1 cDNA fragments by RT-PCR in rat heart. After reverse transcription of total RNA isolated from freshly dissected tissues, transcripts of different {alpha}1 subunits were amplified. The RNA was isolated from rat heart (H), the rat myoblast cell line H9c2 (9), and from rat cerebrum as a positive control (C). No RNA is added in the negative control (-). M, size markers. The expected sizes of the cDNA fragments are shown. (A) Transcripts of L-type {alpha}1 subunits. In rat heart, {alpha}1C (Lane 2) and {alpha}1D transcripts (Lane 7) are detected. The {alpha}1C (Lane 3) but no {alpha}1D cDNA fragment is amplified in H9c2 cells (Lane 8). In cerebrum, both {alpha}1C and {alpha}1D are observed (Lanes 4 and 9). No fragments were detected when RNA was omitted (Lanes 5 and 10). DNA fragments of the housekeeper enzyme hypoxanthine-phosphoribosyl transferase (HPRT) are not amplified without RT (Lanes 13, 15, and 17) but were amplified when the RNA was reverse-transcribed (Lanes 12, 14, and 16). Lane 18, no amplification was performed; Lane 19, complete RT-PCR with the primer for HPRT but without template RNA. The 100-bp markers are loaded in Lanes 1, 6, and 11. (B) Transcripts of non-L-type {alpha}1 subunits. No {alpha}1B fragments are detected in rat heart (Lane 7) or the rat H9c2 cell line (Lane 8). However, {alpha}1A and {alpha}1E are observed in rat heart (Lanes 2 and 14), but only {alpha}1E is amplified as a faint cDNA band in the H9c2 cell line (Lane 13). The 100-bp markers are loaded in Lanes 1, 6, 11, and 16. (C) Transcripts of T-type {alpha}1 subunits. The cDNA fragments of two T-type Ca2+ channels are detected in rat heart, {alpha}1G and {alpha}1H (Lane 1 and Lane 6). Although {alpha}1G is also present in the H9c2 cell line, {alpha}1H is not observed. A faint band of {alpha}1I is seen only in H9c2 cells (Lane 12). The 100-bp markers are loaded in Lanes 5 and 10.

Splice Variant of {alpha}1E Voltage-gated Ca2+ Channel in Heart
Previous studies using RNA extracted from human frozen tissue failed to detect the presence of {alpha}1E transcripts (Vajna et al. 1998 ). In the present study, {alpha}1E transcripts were detected extracting total RNA from rat cardiac tissues immediately after dissection without prior freezing (Fig 2). However, the transcripts of {alpha}1E were still amplified in much lower amounts than from the same amount of total RNA of rat cerebellum (Fig 2A and Fig 2B). The structure of the cardiac {alpha}1E splice variant was deduced by amplifying indicative cDNA fragments of the linker between Domains II and III (II/III loop; see Fig 2A) and the carboxy terminus (Fig 2B). After subcloning and sequencing of fragments from the II/III loop (Fig 2C), the comparison of the {alpha}1E fragment from rat heart showed 100% identity with the cloned rat neuronal and 92% identity with the human neuronal {alpha}1E amino acid sequence when the aligned sequences between insert 1 and insert 2 (Fig 2C) were compared. Insert 1 was absent in the {alpha}1E splice variant detected in heart, whereas insert 2 was present as in the neuronal {alpha}1E. However, the amino acid sequence within insert 2 was highly divergent within its seven amino acids compared to the human {alpha}1E (Fig 2C). Sizes and sequences of the cDNA fragments from both the II/III loop and the carboxy terminus led to the conclusion that the same {alpha}1Ee splice variant is expressed in rat heart as in rat islets of Langerhans and human kidney (Vajna et al. 1998 ).



View larger version (64K):
[in this window]
[in a new window]
 
Figure 2. Identification of {alpha}1E cDNA fragments by RT-PCR and sequencing in rat heart. After reverse transcription of total RNA, transcripts of cardiac {alpha}1E were amplified either from freshly isolated RNA (H1, 0.5 µg; Lanes 2 and 7) or from RNA extracted from frozen tissue (H2, 0.5 µg, Lanes 3 and 8; or H3, 1.0 µg, Lane 4). No DNA fragments were amplified when RNA or reverse transcriptase was omitted. Size markers are loaded in Lanes 1, 5, 6, and 10 (M). (A) Fragments of the II/III loop. Rat cerebellum RNA (Ce, 0.5 µg; Lane 5) was used as a reference and yields three different fragments of 363 bp, 399 bp, and 420 bp, which correspond to cDNA fragments of recently identified {alpha}1E isoforms. The oligonucleotide primers correspond to numbers 3643 and 3642 [Table 1 in Vajna et al. 1998 ]. Arrow indicates the size of the detected fragment in rat heart which was extracted, subcloned, and sequenced (C). (B) Fragments of the carboxy terminus. Rat cerebellum RNA (Ce, 0.5 µg; Lane 9) was used as a reference and yields one major fragment of 498 bp and a minor one of 369 bp. The oligonucleotide primers correspond to numbers 524 and 3250 [Table 1 in Vajna et al. 1998 ]. Arrow indicates the size of the longer cDNA fragment, which was the single fragment beyond detection limit in rat heart (Lanes 7 and 8). (C) Sequence alignment of rat and human {alpha}1E cDNA fragments. The cDNA fragment linking domain II and III was amplified from cardiac total RNA by RT-PCR (rat heart {alpha}1E) and its sequence was aligned to the rat brain (GenBank accession # L15453) and human brain {alpha}1E (GenBank accession # L27745).

Specificity of the Anti-{alpha}1E Serum
After immunoblotting of microsomal membrane proteins, no positive staining was detected in proteins from untransfected HEK-293 cells, whereas in membrane proteins from HEK-2C6 cells a protein of 243 ± 22 kD (n = 4) (Fig 3A) was observed. The apparent size is smaller than the predicted size of the cloned {alpha}1Ed (261,766 Daltons).



View larger version (57K):
[in this window]
[in a new window]
 
Figure 3. Immunoblotting and detection of human {alpha}1Ed in microsomes from stably transfected HEK-293 cells by peptide-specific antibodies. Microsomal membrane proteins (10 µg) from untransfected HEK-293 cells (- -, Lanes 1 and 5), or from cell lines stably transfected with {alpha}1E (E), {alpha}1G (G), or {alpha}1H (H) were analyzed by SDS-PAGE (5% homogeneous gel) and immunoblotting. (A) Specificity of anti-{alpha}1E serum. The polyclonal serum raised against peptide Nast-197, which is part of the carboxy terminal insertion present in the longer {alpha}1E isoforms {alpha}1Ed and {alpha}1Ee (anti-{alpha}1E-spec), was used as the source of primary antibodies in Lanes 1–4. Arrow shows the expected size of the {alpha}1E polypeptide. (B) Effects of temperature on detection of the {alpha}1E subunit. The temperature during electrophoresis was either at 10C (Lanes 1–4) or at 22C (Lanes 5–8). The microsomal membrane proteins were isolated from two different stable cell lines, HEK-2C2 (Lanes 2 and 6) and HEK-2C6 (Lanes 3, 4, 7, and 8), expressing human {alpha}1E. Aliquots of HEK-2C6 membrane proteins had been stored frozen in the presence of SDS denaturing buffer before loading (Lanes 4 and 8). All other aliquots were stored without SDS denaturing buffer and were denatured before use.

The immunodetection of {alpha}1E was dependent on the temperature during SDS-PAGE and on the pretreatment of the membrane proteins before electrophoresis (Fig 3B). When the temperature during electrophoresis was decreased to 10C, the apparent staining of the anti-{alpha}1E-positive band was more intense than after electrophoresis at 22C (Fig 3B; compare Lanes 2–4 with Lanes 6–8). Denaturing of the protein aliquots by SDS denaturing buffer before freezing also diminished the intensity of staining (Fig 3B, Lanes 6 and 8), leading to the conclusion that either the overall integrity of {alpha}1E or the renaturation of the anti-{alpha}1E-spec epitope after denaturing electrophoresis is sensitive to elevated temperature.

The detection of single protein bands in microsomal membranes from stably transfected cells suggests that the polyclonal sera are highly specific for the corresponding {alpha}1 subunit. The reactivity and the specificity of the polyclonal anti-{alpha}1E-spec serum had been evaluated recently by immunocytochemistry (Grabsch et al. 1999 ), which indicates that the anti-1E-spec serum is useful for immunocytochemical detection of longer {alpha}1E splice variants in cell lines and tissues.

Immunoblotting of Microsomal Membrane Proteins from Rat and Guinea Pig Tissues
Microsomal membrane proteins were isolated from rat and guinea pig heart, and from rat cerebrum, the rat insulinoma cell line INS-1, and rat kidney. The solubilized membrane proteins were immunoblotted after PAGE. The anti-{alpha}1E-spec serum stained a protein of 218 ± 6 kD (n = 3) in the cardiac microsomes from both species (Fig 4A). The size of the positive band in rat and guinea pig heart was smaller by 28 kD than the recombinant protein from the stably transfected HEK-293 cell lines. Microsomes of rat cerebrum were used as a reference tissue, because a different {alpha}1E isoform is expressed lacking the anti-{alpha}1E-spec epitope (Pereverzev et al. 1998 ). In this tissue, no positive band was detected by anti-{alpha}1E-spec serum (Fig 4A, Lane 7). However, in INS-1 cells and in kidney, the anti-{alpha}1E-spec serum stained a protein of similar size as in HEK-2C6 cells (Fig 4A). In addition, a shorter positive band was also observed in rat kidney membrane proteins.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 4. Detection of {alpha}1E by immunoblotting of membrane proteins from cell lines, heart, kidney, and cerebrum. (A) Microsomal membrane proteins from untransfected HEK-293 cells (--, 2.5 µg; Lanes 1 and 5), from cell lines stably transfected with {alpha}1E (E, 3 µg; Lane 2), or {alpha}1G (G, 3 µg; Lane 3), from rat heart (rat, 20 µg in Lane 4 or 32 µg in Lane 5), from guinea pig heart (gp, 8 µg; Lane 6), from rat cerebrum (14 µg; Lane 7), from INS-1 cells (INS-1, 25 µg; Lane 8), and from rat kidney (kidn, 50 µg; Lane 9) were analyzed by SDS-PAGE and immunoblotting. Arrow marks the size of the recombinant {alpha}1E protein detected in HEK-2C6 cells (Lane 2). No protein is detected in cerebrum, which mainly expresses {alpha}1E-3. This isoform of {alpha}1E is lacking the epitope for anti-{alpha}1E-spec serum, which was used for the immunodetection. (B) Microsomal membrane proteins from untransfected HEK-293 cells (--, 3 µg; Lanes 1 and 5), from cell lines stably transfected with {alpha}1E (E, 3 µg; Lane 2), or {alpha}1G (G, 3 µg; Lane 3), from rat heart (rat, 20 µg in Lane 4 or 32 µg in Lane 5), from guinea pig heart (gp, 8 µg; Lane 6), and from rat cerebrum (14 µg; Lane 7). Arrow marks the size of the recombinant {alpha}1E protein detected in HEK-2C6 cells (Lane 2). Note that a protein is detected in cerebrum which mainly expresses an {alpha}1E isoform detected by the anti-{alpha}1E-com serum.

The second anti-{alpha}1E serum designed to detect all known {alpha}1E isoforms also stained a 218-kD protein from rat and guinea pig heart (Fig 4B). In addition, a longer {alpha}1E was observed in the heart from guinea pig and in rat cerebrum (Fig 4B). No staining was detected when preimmune serum was used instead of primary antibody.

In rat heart, the molecular size of the putative cardiac {alpha}1E was smaller than the size of the recombinant protein after staining with both anti-{alpha}1E sera (see arrows in Fig 4A and Fig 4B). On the basis of RT-PCR results, a cardiac-specific {alpha}1E splice variant should be truncated by only 2.5 kD in the II/III loop. However, the observed shift was 28 kD and was in the same range as detected for other recombinant {alpha}1 subunits of voltage-gated Ca2+ channels in purified skeletal muscle, cardiac muscle, and neurons (Flockerzi et al. 1986 ; Sieber et al. 1987 ; Schneider and Hofmann 1988 ; DeJongh et al. 1991 ; Florio et al. 1992 ; Hell et al. 1994 ). In conclusion, the {alpha}1Ee isoform, which is structurally different from the major neuronal {alpha}1E isoform, was detected in rat and guinea pig heart as well as in rat INS-1 cells and human kidney, but not in rat cerebrum.

Immunohistochemical Detection of {alpha}1E in Heart
The {alpha}1E splice variant detected in rat and guinea pig heart by RT-PCR and by immunoblotting was predicted to contain the longer carboxy terminus that was originally cloned from human fetal brain (Schneider et al. 1994 ). Hence, immunohistochemical stainings are shown with the anti-{alpha}1E-spec serum throughout this part of the report (Fig 5) but identical results were obtained with the anti-{alpha}1E-com serum and an anti-{alpha}1E serum used for the detection of neuronal {alpha}1E (Volsen et al. 1995 ). Anti-{alpha}1E-spec-positive cells were detected and were scattered throughout the rat heart, in the right atrium (Fig 5A), the left atrium, (Fig 5F), and in both ventricles (Fig 5A and Fig 5B). No staining was observed with the preimmune (Fig 5C and Fig 5D) or the preabsorbed serum (not shown).



View larger version (119K):
[in this window]
[in a new window]
 
Figure 5. Distribution of anti-{alpha}1E-spec positive cells in serial sections from rat heart. Detection of {alpha}1E-positive cells in atrial and ventricular regions. (A) Immunostaining by anti-{alpha}1E-spec (1:100) of cross-sections from right atrium and ventricle. Arrows point to the border between the atrial and ventricular regions leaving the atrium in the upper panel. (B) The anti-{alpha}1E positive cells are shown from the left ventricle at the bottom of the papillar muscle. (C,D) Negative controls of consecutive sections stained with preimmune serum 1:100. Bars = 400 µm. Histochemical detection of Purkinje fibers by HE staining in left atrium and immunohistochemical staining of anti-{alpha}1E-positive myocytes in serial sections of rat heart. (E) Histochemical staining (HE) of serial sections from the left atrium. (F) Immunohistochemical staining using anti-{alpha}1E-spec (1:100) as primary antibody. No staining is detected in the endocardial layer (lowest part of the panel). (G) Negative control, using preimmune serum 1:100. Bars = 50 µm.

To discriminate the conducting muscle fibers from the force-producing muscle fibers, we evaluated hematoxylin–eosin-stained tissue sections (Fig 5E). The cytoplasm of the conducting muscle fibers was less eosinophilic and therefore appeared paler. In addition, it often contained vacuoles or appeared to be "empty," probably due to the fact that conducting muscle fibers have a lower content of regularly organized contractile material and increased numbers of glycogen granules (Viragh et al. 1987 ).

Only the subpopulation of cardiac muscle cells histochemically defined as impulse-conducting fibers (Fig 5E) were anti-{alpha}1E-spec-positive (Fig 5F). A similar staining pattern was observed in serial sections of the right atrium and both ventricles (not shown). No staining was detected with preimmune serum (Fig 5G) or peptide-blocked sera (not shown). Neither preincubation of the sera with 20 µM hemocyanin, the carrier protein for immunization with peptides, nor preincubation with 20 µM cardiac myosin, a major protein of similar size associated with microsomal membranes, blocked the anti-{alpha}1E-spec-positive staining pattern observed in rat heart. Therefore, in rat heart {alpha}1E-expression appears to be restricted to the conducting fibers of the myocardium, including the bundles of His and the Purkinje fibers of the ventricles.

In serial tissue sections, anti-chromogranin A/B (Fig 6A) and anti-ANP antibodies (Fig 6C) were used as markers for endocrine cells, first because we had described {alpha}1E in neuroendocrine cells of the gastrointestinal system (Grabsch et al. 1999 ) and, second, because endocrine functions in the rat heart are assigned to Purkinje fibers (Hansson and Forsgren 1993 ; Benvenuti et al. 1997 ). In the atrium, the anti-chromogranin (Fig 6A) and anti-ANP (Fig 6C) sera stained the same muscle cells as detected by anti-{alpha}1E-spec (Fig 6B). Atrial (Fig 6C) but not ventricular muscle cells (not shown) were stained by the anti-ANP serum. No positive staining was seen when preimmune serum for {alpha}1E was used instead of primary antibody (Fig 6D). We conclude that {alpha}1E is co-expressed with chromogranin A/B in the rat heart and with ANP in the rat atrium.



View larger version (131K):
[in this window]
[in a new window]
 
Figure 6. Comparison of anti-{alpha}1E-spec- and anti-chromogranin-positive cells in serial sections of rat heart. Immunostaining of cardiac muscle cells by anti-chromogranin, anti-ANP, anti-{alpha}1E sera in serial sections. (A) Immunohistochemical detection of anti-chromogranin A/B-positive cells (rat-specific serum, 1:50). (B) Immunostaining of anti-{alpha}1E positive cells by anti-{alpha}1E-spec (1:300). (C) Immunostaining of anti-ANP positive cells by anti-ANP (1:200). (D) No staining was observed in negative controls using preimmune serum. Bars = 100 µm. Histochemical staining of the conduction system in the rat truncus atrioventricularis. (E) Histochemical (HE) staining of serial sections containing part of the impulse conduction system. (F) Immunohistochemical staining of a consecutive section using anti-{alpha}1E-spec serum as primary antibody (1:100). Bars = 200 µm.

In cross-sections through the bundle of His (truncus atrioventricularis), bundles of conducting fibers (Fig 6E, central part) reacted strongly with the anti-{alpha}1E-spec serum. The immunohistochemically defined distribution of {alpha}1E led us to suggest that it may be involved in both endocrine and conducting functions in rat heart.

To confine and to confirm the expression of {alpha}1E in muscle cells related to the endocrine system of the rat heart, expression of the {alpha}1E subunit (Fig 7B and Fig 7C), ANP (Fig 7A), and chromogranin (Fig 7D and Fig 7E) was investigated in the rat cardiomyoblast cell line H9c2. These cells were positively stained by anti-{alpha}1E-spec, anti-ANP, and anti-chromogranin, but not by the preimmune serum (Fig 7F).



View larger version (125K):
[in this window]
[in a new window]
 
Figure 7. Immunodetection of anti-{alpha}1E-spec-positive cells in the rat H9c2 cell line and in human kidney. Immunocytochemical staining of H9c2 cells. (A) Staining by anti-ANP (1:200). Bar = 100 µm. (B) Staining by anti-{alpha}1E-spec serum (1:300). Bar = 400 µm. (C) Staining by anti-{alpha}1E-spec serum (1:300). Bar = 100 µm. (D) Staining by anti-chromogranin A/B (1:50). Bar = 400 µm. (E) Staining by anti-chromogranin A/B (1:50). Bar = 100 µm. (F) Staining by preimmune serum (1:300) from the rabbit immunized with the peptide Nast-197 (yielding anti-{alpha}1E-spec serum). Bar = 400 µm. Immunocytochemical staining of human kidney. (G) Staining of distal tubules (D) in the proximity of a glomerulus (Gl). Only faint if any staining is seen in cross-sections of proximal tubules (P). (H) Negative control using preimmune serum. Bars = 50 µm.

Immunohistochemical Detection of {alpha}1E in Kidney
The secretion of the peptide hormone urodilatin in kidney serves similar functions as the ANP secretion in heart (Forssmann et al. 1998 ; Herten et al. 1998 ). Immunohistochemical staining of human kidney by anti-{alpha}1E-spec serum revealed a strong positive reaction in distal tubules and a weaker one in proximal tubules (Fig 7G). No staining was observed in the glomerulus (Fig 7G) or in the negative control incubated with preimmune serum (Fig 7H). Identical patterns were observed with the anti-{alpha}1E-com or the serum received from Dr. S.G. Volsen (Volsen et al. 1995 ). Similar immunohistochemical results were obtained from rat kidney (not shown). The staining of human tissue is shown because of the easier discrimination between proximal and distal tubules and the well-known functions related to individual regions of the kidney.

In summary, {alpha}1E was detected by RT-PCR in rat and human kidney (Vajna et al. 1998 ), by immunoblotting in rat kidney (see Fig 4), and by immunohistochemical staining mainly in the distal tubules of human kidney. This leads to the conclusion that {alpha}1E might be related to functions typical for the distal tubules in kidney.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Transcripts of Voltage-gated Ca2+ Channels in Rat Heart
In heart, T- and L-type voltage-gated Ca2+ channels are involved in pacemaker activity and excitation–contraction coupling. After the reported cloning of 10 different {alpha}1 subunits, a more detailed analysis by RT-PCR reveals that at least transcripts of two different L-type ({alpha}1C, {alpha}1D), two non-L-type ({alpha}1A, {alpha}1Ee), and two T-type Ca2+ channels ({alpha}1G, {alpha}1H) are amplified from rat heart. In the present study, one of them, a specific splice variant of {alpha}1E, was characterized in greater detail. The expression of {alpha}1E in rat and guinea pig heart was also investigated at the protein level because an IGF-induced T-type Ca2+ current was reduced in rat atrial myocytes after application of {alpha}1E-specific antisense oligonucleotides (Piedras-Renteria et al. 1997 ).

Initially, we failed to amplify {alpha}1E transcripts from frozen human heart (Vajna et al. 1998 ). However, using rats, in which RNA can be extracted directly from freshly isolated heart without prior freezing of tissue, we were able to detect faint bands amplified from the regions indicative for one of the several splice variants of {alpha}1E. The most indicative cDNA fragments (II/III loop) were subcloned and identified by sequencing to be related to the same {alpha}1Ee isoform as found in islets of Langerhans and kidney (Vajna et al. 1998 ), which is in line with recent results (Mitchell et al. 1999 ).

The {alpha}1Ee isoform is found in neuroendocrine tissues and is also expressed in cerebellum. Its tissue-specific expression in islets of Langerhans, endocrine cells of the gastrointestinal tract (Grabsch et al. 1999 ), anterior pituitary gland, thyroid, parathyroid glands, and adrenal gland (unpublished data), as well as in kidney (Vajna et al. 1998 ), might point to a specific function of {alpha}1E within endocrine signaling pathways.

The primary structure of the cardiac {alpha}1Ee isoform shows major differences from the cloned neuronal {alpha}1Ed (Schneider et al. 1994 ) in the II/III loop and from the carboxy terminus of cloned {alpha}1E-3 (Williams et al. 1994 ). Within the II/III loop, insert 1 probably represents a duplication of an adjacent arginine-rich sequence: a stretch of 19 amino acids is lacking in {alpha}1Ee and starts at position aa 749 with RERRRRHHMS, resembling the adjacent sequence at position aa 768 of human {alpha}1E, RERRRRHHMS. On the other side, this sequence shows homology to a skeletal muscle {alpha}1S sequence, EERKRR.KMS, which is also located in the II/III loop of {alpha}1S and is believed to be involved in excitation–contraction coupling (El-Hayek and Ikemoto 1998 ). A similar functional interaction of the II/III loop with synaptic vesicle proteins might occur for {alpha}1E during excitation–secretion coupling, as was shown for {alpha}1A, {alpha}1B (Rettig et al. 1996 ), and {alpha}1C (Wiser et al. 1999 ; Sheng et al. 1996 ).

The carboxy terminus of {alpha}1Ed and {alpha}1Ee contains an insertion of 43 amino acids of yet unknown function. After deletion of these 43 amino acids, the basic electrophysical properties are not changed in the deletion mutant compared to the wild-type {alpha}1Ed, as was shown after stable transfection in HEK293 cells (Pereverzev et al. 1998 ).

Detection of {alpha}1E by Immunoblotting
The apparent molecular size of {alpha}1E was determined by immunoblotting of microsomal membrane proteins from several tissues, including rat and guinea pig heart. The cardiac protein, which was stained by two different anti-{alpha}1E sera after immunoblotting, is truncated to some extent and its apparent size is smaller by 28 ± 16 kD than the reference protein. A shift of only 2.5 kD was expected because the recombinant {alpha}1Ed contains the 19-aa insert 1 of the II/III loop. Because both sera detected {alpha}1E in rat and guinea pig heart, the {alpha}1E subunit might be truncated downstream of the epitope recognized by anti-{alpha}1E-spec. The truncation might be caused either by posttranslational processing or by in vitro proteolysis during isolation of the membrane proteins, or by both.

Detection of {alpha}1E in Fibers Related to the Impulse-conducting System of the Rat Heart and in the Distal Tubules of Human Kidney
Three different sera against {alpha}1E were used during the immunohistochemical investigation of anti-{alpha}1E positive muscle cells in the rat heart. Two sera were created by linking peptides to hemocyanin (Pereverzev et al. 1998 ) and one was independently created by fusing a peptide to gluthathion S-transferase (Volsen et al. 1995 ). Identical results were obtained with all three {alpha}1E-directed sera. Anti-{alpha}1E-positive muscle cells were found in the right and left atrium and also in both ventricles. The cell morphology itself and the HE staining of Purkinje fibers clearly demonstrated that the anti-{alpha}1E-positive muscle cells are part of the impulse-conducting system.

Several findings support our interpretation of an endocrine function for {alpha}1E in rat heart. First, the distribution of anti-{alpha}1E-positive cells in heart closely resembles the distribution of chromogranin A/B, which is co-localized with ANP (Steiner et al. 1990 ). Second, cardiac peptide hormones such as ANP are widely expressed in the conducting system of rat (Cantin et al. 1989 ), bovine (Hansson and Forsgren 1993 , Hansson and Forsgren 1995 ), and human (Benvenuti et al. 1997 ). They are detected in atrial and ventricular walls (Toshimori et al. 1988 ). Therefore, we favor the idea that {alpha}1E in atrium as well as in ventricle of rat and guinea pig heart might participate in triggering the secretion of peptide hormones, comparable to its recently deduced function in islets of Langerhans and endocrine cells of the gastrointestinal system (Grabsch et al. 1999 ).

Before the cardiac expression of {alpha}1E had been investigated, its distribution in human kidney had been analyzed. The fact that anti-{alpha}1E-positive cells were found mainly in the distal tubules could also be related to an endocrine function of {alpha}1E for the secretion of urodilatin in kidney. The renal natriuretic peptide urodilatin is synthesized in the distal tubule region (Meyer et al. 1996 ; Forssmann et al. 1998 ). It may be secreted and transported as a paracrine factor to the collecting duct, where it suppresses sodium reabsorption by stimulating guanylyl cyclase (Goetz 1991 ; Forssmann et al. 1998 ; Herten et al. 1998 ).

Other functions, such as impulse generation and pacemaking, might also be related to the increasing number of putative T-type Ca2+ channels identified in heart. Recent reports have shown that T-type Ca2+ currents not only contribute to pacemaking (Irisawa et al. 1993 ) but also might be able to more directly trigger Ca2+ release from sarcoplasmic reticulum in guinea pig (Sipido et al. 1998 ) and canine Purkinje myocytes (Zhou and January 1998 ). In this regard, it might be interesting to investigate the IGF-induced T-type currents (Piedras-Renteria et al. 1997 ), which perhaps are involved in peptide hormone secretion during differentiation of the atrial myocytes in culture (Xu and Best 1992 ). Future experiments might elucidate such a function using {alpha}1E-specific peptide toxins (Newcomb et al. 1998 ).


  Footnotes

1 These authors contributed equally to the work.


  Acknowledgments

Supported by the Köln Fortune Program/Faculty of Medicine, University of Köln, and the Center of Molecular Medicine Köln/Zentrum für Molekulare Medizin Köln (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Förderkennzeichen 01 KS 9502).

We thank Prof Dr J. Hescheler for providing us his help and excellent opportunities in his facilities for research, and Dr R.E. Beattie, Dr P.J. Craig, and Dr S.G. Volsen (Lilly Company; Windlesham, London) for a third serum against {alpha}1E. We acknowledge the careful reading of the manuscript by Prof Dr E. Perez–Reyes (Charlottesville, VA). We thank Ms M. Chludek (Leverkusen, Germany) for her help during preparation of serial sections, and Ms R. Clemens and Ms S. Schulze for their permanent technical assistance.

Received for publication February 4, 2000; accepted February 9, 2000.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Asfari M, Janjic D, Med P, Li G, Halban PA, Wollheim CB (1992) Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130:167-178[Abstract]

Bean BP (1985) Two kinds of calcium channels in canine atrial cells. J Gen Physiol 86:1-30[Abstract]

Bean BP (1989) Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340:153-156[Medline]

Bers DR, Perez–Reyes E (1999) Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res 42:339-360[Medline]

Benvenuti LA, Aiello VD, Higuchi MdL, Palomino SA (1997) Immunohistochemical expression of atrial natriuretic peptide (ANP) in the conducting system and internodal atrial myocardium of human hearts. Acta Histochem 99:187-193[Medline]

Cantin M, Thibault G, Haile–Meskel H, Ding J, Milne RW, Ballak M, Charbonneau C, Nemer M, Drouin J, Garcia R, Genest J (1989) Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles. Cell Tissue Res 256:309-325[Medline]

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159[Medline]

Cribbs LL, Lee J-H, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, Perez–Reyes E (1998) Cloning and characterization of {alpha}1H from human heart, a member of the T-type calcium channel gene family. Circ Res 83:103-109[Abstract/Free Full Text]

DeJongh KS, Warner C, Colvin AA, Catterall WA (1991) Characterization of the two size forms of the alpha 1 subunit of skeletal muscle L-type calcium channels. Proc Natl Acad Sci USA 88:10778-10782[Abstract]

El-Hayek R, Ikemoto N (1998) Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor {alpha}1 subunit required for activation of skeletal muscle-type excitation-contraction coupling. Biochemistry 37:7015-7020[Medline]

Ferry DR, Goll A, Glossmann H (1987) Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]-azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand. Biochem J 243:127-135[Medline]

Flockerzi V, Oeken HJ, Hofmann F (1986) Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes. Eur J Biochem 161:217-224[Abstract]

Florio V, Striessnig J, Catterall WA (1992) Purification and reconstitution of skeletal muscle calcium channels. Methods Enzymol 207:529-546[Medline]

Forssmann W-G, Richter R, Meyer M (1998) The endocrine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system. Histochem Cell Biol 110:335-357[Medline]

Goetz KL (1991) Renal natriuretic peptide (urodilatin?) and atriopeptin: evolving concepts. Am J Physiol 261:F921-932[Abstract/Free Full Text]

Grabsch H, Pereverzev A, Weiergräber M, Schramm M, Henry M, Vajna R, Beattie RE, Volsen SG, Klöckner U, Hescheler J, Schneider T (1999) Immunohistochemical detection of {alpha}1E voltage-gated Ca2+ channel isoforms in cerebellum, INS-1 cells, and neuroendocrine cells of the digestive system. J Histochem Cytochem 47:981-993[Abstract/Free Full Text]

Haase H, Striessnig J, Holtzhauer M, Vetter R, Glossmann H (1991) A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart. Eur J Pharmacol 207:51-59[Medline]

Handrock R, Schröder F, Pereverzev A, Schneider T, Cribbs LL, Perez–Reyes E, Herzig S (1999) Single-channel properties of {alpha}1E- and {alpha}1H-calcium channels compared to cardiac T-type calcium channels. Biophys J 76:A409

Hansson M, Forsgren S (1993) Presence of immunoreactive atrial natriuretic peptide in nerve fibres and conduction cells in the conduction system of the bovine heart. Anat Embryol 188:331-337[Medline]

Hansson M, Forsgren S (1995) Immunoreactive atrial and brain natriuretic peptides are co-localized in Purkinje fibres but not in the innervation of the bovine heart conduction system. Histochem J 27:222-230[Medline]

Hell JW, Westenbroek RE, Elliott EM, Catterall WA (1994) Differential phosphorylation, localization, and function of distinct {alpha}1 subunits of neuronal calcium channels. Two size forms for class B, C, and D {alpha}1 subunits with different COOH-termini. Ann NY Acad Sci 747:282-293[Medline]

Herten M, Lenz W, Gerzer R, Drummer C (1998) The renal natriuretic peptide urodilatin is present in human kidney. Nephrol Dial Transplant 13:2529-2535[Abstract]

Hess P (1990) Calcium channels in vertebrate cells. Annu Rev Neurosci 13:337-356[Medline]

Irisawa H, Brown HF, Giles W (1993) Cardiac pacemaking in the sinoatrial node. Physiol Rev 73:197-227[Free Full Text]

Kawano S, DeHaan RL (1991) Developmental changes in the calcium currents in embryonic chick ventricular myocytes. J Membr Biol 120:17-28[Medline]

Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klöckner U, Schneider T, Perez–Reyes E (1999) Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19:1912-1921[Abstract/Free Full Text]

Mehrke G, Pereverzev A, Grabsch H, Hescheler J, Schneider T (1997) Receptor mediated modulation of recombinant neuronal class E calcium channels. FEBS Lett 408:261-270[Medline]

Merrifield B (1986) Solid phase synthesis. Science 232:241-247

Meyer M, Stief CG, Becker AJ, Truss MC, Taher A, Jonas U, Forssmann W-G (1996) The renal paracrine peptide system—possible urologic implications of urodilatin. World J Urol 14:375-379[Medline]

Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, Narumiya S, Numa S (1989) Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340:230-233[Medline]

Mitchell JW, Larsen JK, Best PM (1999) Sequence of E-type calcium channel gene isoforms from atrial myocytes and the cellular distribution of their protein products. Biophys J 76:A91

Mittman S, Guo J, Emerick MC, Agnew WS (1999) Structure and alternative splicing of the gene encoding {alpha}1I, a human brain T calcium channel {alpha}1 subunit. Neurosci Lett 269:121-124[Medline]

Nakashima YM, Todorovic SM, Pereverzev A, Hescheler J, Schneider T, Lingle CJ (1998) Properties of Ca2+ currents arising from human {alpha}1E and {alpha}1Eß3 constructs expressed in HEK293 cells: physiology, pharmacology, and comparison to native T-type Ca2+ currents. Neuropharmacology 37:957-972[Medline]

Newcomb R, Szoke B, Palma A, Wang G, Chen XH, Hopkins W, Cong R, Miller J, Urge L, Tarczy–Hornoch K, Loo JA, Dooley DJ, Nadasdi L, Tsien RW, Lemos J, Miljanich G (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 37:15353-15362[Medline]

Nilius B (1998) T-type calcium channels in myocardial cells. In Tsien RW, Clozel J-P, Nargeot J, eds. Low-Voltage-Activated T-type Calcium Channels. Tattenhall: Adis International, 1, 6-28

Nilius B, Hess P, Lansman JB, Tsien RW (1985) A novel type of cardiac calcium channel in ventricular cells. Nature 316:443-446[Medline]

Pereverzev A, Klöckner U, Henry M, Grabsch H, Vajna R, Olyschläger S, Viatchenko–Karpinski S, Schröder R, Hescheler J, Schneider T (1998) Structural diversity of the voltage-dependent Ca2+ channel {alpha}1E subunit. Eur J Neurosci 10:916-925[Medline]

Perez–Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, Lee J-H (1998) Molecular characterization of a neuronal low voltage-activated T-type calcium channel. Nature 391:896-900[Medline]

Piedras–Rentería ES, Chen CC, Best PM (1997) Antisense oligonucleotides against rat brain alpha1E DNA and its atrial homologue decrease T-type calcium current in atrial myocytes. Proc Natl Acad Sci USA 94:14936-14941[Abstract/Free Full Text]

Randall AD, Tsien RW (1997) Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels. Neuropharmacology 36:879-893[Medline]

Rettig J, Sheng ZH, Kim DK, Hodson CD, Snutch TP, Catterall WA (1996) Isoform-specific interaction of the {alpha}1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25. Proc Natl Acad Sci USA 93:7363-7368[Abstract/Free Full Text]

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467[Abstract]

Schneider T, Hofmann F (1988) The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Eur J Biochem 174:369-375[Abstract]

Schneider T, Regulla S, Nastainczyk W, Hofmann F (1992) Purification and structure of L-type calcium channels. In Longstaff A, Revest P, eds. Methods in Molecular Neurobiology. Vol 13. Protocols in Molecular Neurobiology. Totowa, NJ: Humana Press, 273-286

Schneider T, Wei X, Olcese R, Costantin JL, Neely A, Palade P, Perez–Reyes E, Qin N, Zhou J, Crawford GD, Smith RG, Appel SH, Stefani E, Birnbaumer L (1994) Molecular analysis and functional expression of the human type E {alpha}1 subunit. Receptors Channels 2:255-270[Medline]

Seisenberger C, Welling A, Pfeifer A, Klugbauer N, Hofmann F (1999) Disruption of the calcium channel {alpha}1C-gene by gene targeting show a beating heart later than day 9.5 p.c. Naunyn Schmiedeberg Arch Pharmacol 359:R66

Sheng Z-H, Rettig J, Cook T, Catterall WA (1996) Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379:451-454[Medline]

Sieber M, Nastainczyk W, Zubor V, Wernet W, Hofmann F (1987) The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel. Eur J Biochem 167:117-122[Abstract]

Sipido KR, Carmeliet E, Van de Werf F (1998) T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes. J Physiol (Lond) 508:439-451[Abstract/Free Full Text]

Steiner H-J, Weiler R, Ludescher C, Schmid KW, Winkler H (1990) Chromogranins A and B are co-localized with atrial natriuretic peptides in secretory granules of rat heart. J Histochem Cytochem 38:845-850[Abstract]

Toshimori H, Toshimori K, Oura C, Matsuo H, Matsukura S (1988) The distribution of atrial natriuretic polypeptide (ANP)-containing cells in the adult rat heart. Anat Embryol 177:477-484[Medline]

Trautwein W, Kameyama M, Hescheler J, Hofmann F (1986) Cardiac calcium channels and their transmitter modulation. Prog Zool 33:163-182

Tytgat J, Nilius B, Vereecke J, Carmeliet E (1988) The T-type Ca channel in guinea-pig ventricular myocytes is insensitive to isoproterenol. Pflugers Arch 411:704-706[Medline]

Vajna R, Schramm M, Pereverzev A, Arnhold S, Grabsch H, Klöckner U, Perez–Reyes E, Hescheler J, Schneider T (1998) New isoform of the neuronal Ca2+ channel {alpha}1E subunit in islets of Langerhans, and kidney. Distribution of voltage-gated Ca2+ channel {alpha}1 subunits in cell lines and tissues. Eur J Biochem 257:274-285[Abstract]

Vassort G, Alvarez J (1994) Cardiac T-type calcium current: pharmacology and roles in cardiac tissues. J Cardiovasc Electrophysiol 5:376-393[Medline]

Viragh S, Stoeckel ME, Porte A (1987) Light and electron microscopic structure of the cardiac Purkinje fibers—review. Physiol Bohemoslov 36:233-242[Medline]

Volsen SG, Day NC, McCormack AL, Smith W, Craig PJ, Beattie R, Ince PG, Shaw PJ, Ellis SB, Gillespie A, Harpold MM, Lodge D (1995) The expression of neuronal voltage-dependent calcium channels in human cerebellum. Mol Brain Res 34:271-282[Medline]

Williams ME, Marubio LM, Deal CR, Hans M, Brust PF, Philipson LH, Miller RJ, Johnson EC, Harpold MM, Ellis SB (1994) Structure and functional characterization of neuronal {alpha}1E calcium channel subtypes. J Biol Chem 269:22347-22357[Abstract/Free Full Text]

Wiser O, Trus M, Hernández A, Renström E, Barg S, Rorsman P, Atlas D (1999) The voltage-sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci USA 96:248-253[Abstract/Free Full Text]

Wyatt CN, Campbell V, Brodbeck J, Brice NL, Page KM, Berrow NS, Brickley K, Terracciano CMN, Naqvi RU, MacLeod KT, Dolphin AC (1997) Voltage-dependent binding and calcium channel current inhibition by an anti-{alpha}1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes. J Physiol (Lond) 502:307-319[Abstract]

Xu X, Best PM (1992) Postnatal changes in T-type calcium current density in rat atrial myocytes. J Physiol 454:657-672[Abstract]

Zhou ZF, January CT (1998) Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells. Biophys J 74:1830-1839[Abstract/Free Full Text]