Abstract
The calcium channel α1E 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 α1E 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 α1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to α1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of α1E 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 α1E 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 α1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of α1E 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.
Keywords
C
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 (α1G, α1H, and α1I) 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 (α1G, α1H) 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 α1E-induced Ca2+ inward currents, because antisense oligonucleotides against α1E mRNA inhibited the IGF-induced T-type current (Piedras-Rentería 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 α1E is not considered a typical T-type current (Randall and Tsien 1997; Nakashima et al. 1998), the immunodetection of α1E as a method independent of RT-PCR amplification (Piedras-Rentería et al. 1997; Mitchell et al. 1999) should give additional insight into a possible physiological role for α1E in heart. Therefore, the distribution of α1E was compared with the expression of neuroendocrine markers (chromogranin A/B) in the myocardium. Furthermore, the distribution of α1E 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
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 α1 subunits from high-voltage-activated Ca2+ channels and for the housekeeping enzyme hypoxanthine phosphoribosyltransferase have been summarized recently (Vajna et al. 1998). The rat α1G cDNA fragment was amplified using the primer Gfrwsp, 5′-AGCCCCGGTGGTTTCTTCTA-3′ (nt 650-670) of rat α1G cDNA sequence (GenBank AF027984; Bethesda, MD) as forward and Grevsp, 5′-TGAGCGGTCGCAGCACAC-3′ as reverse primer (nt 1047-1030). The rat α1H cDNA fragment was amplified using oligo-452341, 5′-AGGAGGCTCGGCGCCGG-3′ (homologue to human α1H, AF051946, nt 4777-4793) as forward and oligo-452340, 5′-GGATAGGAGGACGATGGCCAA-3′ (homologue to human α1H, nt 5131-5150) as reverse primer. The rat α1I cDNA fragment was amplified using Ifrwis, 5′-GCTGCGGCGCCTGGAAAAGAA-3′ (nt 4501-4521) of rat α1I cDNA sequence (GenBank AF086827) as forward and Irevis, 5′-GCCCATGCACGGACAGCAGCACAAT-3′ as reverse primer (nt 4838-4814). The annealing temperatures were 57C for α1G and α1H, and 60C for α1I 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 α1E sequences were coupled to hemocyanin and used for immunization of rabbits. Peptide Nast-195 corresponds to a common sequence in all published α1E isoforms and peptide Nast-197 is part of a 43-amino-acid extension in the carboxy terminus of the fetal brain-derived α1Ed isoform (Schneider et al. 1994; Pereverzev et al. 1998), which is also located in two other deduced longer α1E isoforms, α1Ee and α1Ef (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 α1E 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-α1E sera were tested by immunoblots of microsomal membrane proteins from stably transfected HEK-293 cells, which express the human α1Ed (cell line HEK-2C6 or HEK-2C2), or the deletion mutant α1Ed-CDEL (cell line HEK-α1E-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-α1E.
In some experiments, the purified anti-α1E serum against an α1E fusion protein (Volsen et al. 1995) and a monoclonal antibody against α1E, 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-α1E-spec serum was not observed with the shorter splice variant α1E-CDEL (Nakashima et al. 1998; Pereverzev et al. 1998) nor with α1G-, α1H-, or α1I-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 α1E (Schneider et al. 1994).
Results
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 α1 subunits either from rat cerebrum as a reference or from rat heart cDNA (Figure 1). The transcripts of two L-type α1 subunits, α1C and α1D, were detected. In addition, fragments of two non-L-type (α1A and 1E) and two T-type subunits (α1G and α1H) were amplified (Figure 1). In the cardiac rat cell line H9c2, neither α1D, α1A, nor α1H was observed, but low amounts of α1I were detected (Figure 1). For α1C and α1H, additional splice variants were deduced by the size of the amplified fragment. No transcripts were amplified with primer pairs for α1S in adult rat heart (not shown).
Splice Variant of α1E Voltage-gated Ca2+ Channel in Heart
Previous studies using RNA extracted from human frozen tissue failed to detect the presence of α1E transcripts (Vajna et al. 1998). In the present study, α1E transcripts were detected extracting total RNA from rat cardiac tissues immediately after dissection without prior freezing (Figure 2). However, the transcripts of α1E were still amplified in much lower amounts than from the same amount of total RNA of rat cerebellum (Figures 2A and 2B). The structure of the cardiac α1E splice variant was deduced by amplifying indicative cDNA fragments of the linker between Domains II and III (II/III loop; see Figure 2A) and the carboxy terminus (Figure 2B). After subcloning and sequencing of fragments from the II/III loop (Figure 2C), the comparison of the α1E fragment from rat heart showed 100% identity with the cloned rat neuronal and 92% identity with the human neuronal α1E amino acid sequence when the aligned sequences between insert 1 and insert 2 (Figure 2C) were compared. Insert 1 was absent in the α1E splice variant detected in heart, whereas insert 2 was present as in the neuronal α1E. However, the amino acid sequence within insert 2 was highly divergent within its seven amino acids compared to the human α1E (Figure 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 α1Ee splice variant is expressed in rat heart as in rat islets of Langerhans and human kidney (Vajna et al. 1998).
Detection of α1 cDNA fragments by RT-PCR in rat heart. After reverse transcription of total RNA isolated from freshly dissected tissues, transcripts of different α1 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. (
Specificity of the Anti-α1E 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) (Figure 3A) was observed. The apparent size is smaller than the predicted size of the cloned α1Ed (261,766 Daltons).
The immunodetection of α1E was dependent on the temperature during SDS-PAGE and on the pretreatment of the membrane proteins before electrophoresis (Figure 3B). When the temperature during electrophoresis was decreased to 10C, the apparent staining of the anti-α1E-positive band was more intense than after electrophoresis at 22C (Figure 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 (Figure 3B, Lanes 6 and 8), leading to the conclusion that either the overall integrity of α1E or the renaturation of the anti-α1E-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 α1 subunit. The reactivity and the specificity of the polyclonal anti-α1E-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 α1E 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-α1E-spec serum stained a protein of 218 ± 6 kD (n = 3) in the cardiac microsomes from both species (Figure 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 α1E isoform is expressed lacking the anti-α1E-spec epitope (Pereverzev et al. 1998). In this tissue, no positive band was detected by anti-α1E-spec serum (Figure 4A, Lane 7). However, in INS-1 cells and in kidney, the anti-α1E-spec serum stained a protein of similar size as in HEK-2C6 cells (Figure 4A). In addition, a shorter positive band was also observed in rat kidney membrane proteins.
Identification of α1E cDNA fragments by RT-PCR and sequencing in rat heart. After reverse transcription of total RNA, transcripts of cardiac α1E 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). (
The second anti-α1E serum designed to detect all known α1E isoforms also stained a 218-kD protein from rat and guinea pig heart (Figure 4B). In addition, a longer α1E was observed in the heart from guinea pig and in rat cerebrum (Figure 4B). No staining was detected when preimmune serum was used instead of primary antibody.
In rat heart, the molecular size of the putative cardiac α1E was smaller than the size of the recombinant protein after staining with both anti-α1E sera (see arrows in Figures 4A and 4B). On the basis of RT-PCR results, a cardiac-specific α1E 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 α1 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 α1Ee isoform, which is structurally different from the major neuronal α1E 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 α1E in Heart
The α1E 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-α1E-spec serum throughout this part of the report (Figure 5) but identical results were obtained with the anti-α1E-com serum and an anti-α1E serum used for the detection of neuronal α1E (Volsen et al. 1995). Anti-α1E-spec-positive cells were detected and were scattered throughout the rat heart, in the right atrium (Figure 5A), the left atrium, (Figure 5F), and in both ventricles (Figures 5A and 5B). No staining was observed with the preimmune (Figures 5C and 5D) or the preabsorbed serum (not shown). To discriminate the conducting muscle fibers from the force-producing muscle fibers, we evaluated hematoxylin-eosin-stained tissue sections (Figure 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).
Immunoblotting and detection of human α1Ed 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 α1E (E), α1G (G), or α1H (H) were analyzed by SDS-PAGE (5% homogeneous gel) and immunoblotting. (
Detection of α1E by immunoblotting of membrane proteins from cell lines, heart, kidney, and cerebrum. (
Distribution of anti-α1E-spec positive cells in serial sections from rat heart. Detection of α1E-positive cells in atrial and ventricular regions. (
Only the subpopulation of cardiac muscle cells histochemically defined as impulse-conducting fibers (Figure 5E) were anti-α1E-spec-positive (Figure 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 (Figure 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-α1E-spec-positive staining pattern observed in rat heart. Therefore, in rat heart α1E-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 (Figure 6A) and anti-ANP antibodies (Figure 6C) were used as markers for endocrine cells, first because we had described α1E 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 (Figure 6A) and anti-ANP (Figure 6C) sera stained the same muscle cells as detected by anti-α1E-spec (Figure 6B). Atrial (Figure 6C) but not ventricular muscle cells (not shown) were stained by the anti-ANP serum. No positive staining was seen when preimmune serum for α1E was used instead of primary antibody (Figure 6D). We conclude that α1E is co-expressed with chromogranin A/B in the rat heart and with ANP in the rat atrium.
In cross-sections through the bundle of His (truncus atrioventricularis), bundles of conducting fibers (Figure 6E, central part) reacted strongly with the anti-α1E-spec serum. The immunohistochemically defined distribution of α1E 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 α1E in muscle cells related to the endocrine system of the rat heart, expression of the α1E subunit (Figures 7B and 7C), ANP (Figure 7A), and chromogranin (Figures 7D and 7E) was investigated in the rat cardiomyoblast cell line H9c2. These cells were positively stained by anti-α1E-spec, anti-ANP, and anti-chromogranin, but not by the preimmune serum (Figure 7F).
Immunohistochemical Detection of α1E 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-α1E-spec serum revealed a strong positive reaction in distal tubules and a weaker one in proximal tubules (Figure 7G). No staining was observed in the glomerulus (Figure 7G) or in the negative control incubated with preimmune serum (Figure 7H). Identical patterns were observed with the anti-α1E-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, α1E was detected by RT-PCR in rat and human kidney (Vajna et al. 1998), by immunoblotting in rat kidney (see Figure 4), and by immunohistochemical staining mainly in the distal tubules of human kidney. This leads to the conclusion that α1E might be related to functions typical for the distal tubules in kidney.
Discussion
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 α1 subunits, a more detailed analysis by RT-PCR reveals that at least transcripts of two different L-type (α1C, α1D), two non-L-type (α1A, α1Ee), and two T-type Ca2+ channels (α1G, α1H) are amplified from rat heart. In the present study, one of them, a specific splice variant of α1E, was characterized in greater detail. The expression of α1E 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 α1E-specific antisense oligonucleotides (Piedras-Rentería et al. 1997).
Comparison of anti-α1E-spec- and anti-chromogranin-positive cells in serial sections of rat heart. Immunostaining of cardiac muscle cells by anti-chromogranin, anti-ANP, anti-α1E sera in serial sections. (
Initially, we failed to amplify α1E 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 α1E. The most indicative cDNA fragments (II/III loop) were subcloned and identified by sequencing to be related to the same α1Ee 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 α1Ee 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 α1E within endocrine signaling pathways.
The primary structure of the cardiac α1Ee isoform shows major differences from the cloned neuronal α1Ed (Schneider et al. 1994) in the II/III loop and from the carboxy terminus of cloned α1E-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 α1Ee and starts at position aa 749 with RERRRRHHMS, resembling the adjacent sequence at position aa 768 of human α1E, RERRRRHHMS. On the other side, this sequence shows homology to a skeletal muscle α1S sequence, EERKRR.KMS, which is also located in the II/III loop of α1S 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 α1E during excitation-secretion coupling, as was shown for α1A, α1B (Rettig et al. 1996), and α1C (Wiser et al. 1999; Sheng et al. 1996).
The carboxy terminus of α1Ed and α1Ee 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 α1Ed, as was shown after stable transfection in HEK293 cells (Pereverzev et al. 1998).
Detection of α1E by Immunoblotting
The apparent molecular size of α1E 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-α1E 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 α1Ed contains the 19-aa insert 1 of the II/III loop. Because both sera detected α1E in rat and guinea pig heart, the α1E subunit might be truncated downstream of the epitope recognized by anti-α1E-spec. The truncation might be caused either by posttranslational processing or by in vitro proteolysis during isolation of the membrane proteins, or by both.
Immunodetection of anti-α1E-spec-positive cells in the rat H9c2 cell line and in human kidney. Immunocytochemical staining of H9c2 cells. (
Detection of α1E in Fibers Related to the Impulse-conducting System of the Rat Heart and in the Distal Tubules of Human Kidney
Three different sera against α1E were used during the immunohistochemical investigation of anti-α1E 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 α1E-directed sera. Anti-α1E-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-α1E-positive muscle cells are part of the impulse-conducting system.
Several findings support our interpretation of an endocrine function for α1E in rat heart. First, the distribution of anti-α1E-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 1993,1995), and human (Benvenuti et al. 1997). They are detected in atrial and ventricular walls (Toshimori et al. 1988). Therefore, we favor the idea that α1E 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 α1E had been investigated, its distribution in human kidney had been analyzed. The fact that anti-α1E-positive cells were found mainly in the distal tubules could also be related to an endocrine function of α1E 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-Rentería 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 α1E-specific peptide toxins (Newcomb et al. 1998).
Footnotes
Acknowledgements
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 α1E. 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.