Molecular diversity of KV alpha - and beta -subunit expression in canine gastrointestinal smooth muscles

Anne Epperson1, Helena P. Bonner1, Sean M. Ward1, William J. Hatton1, Karri K. Bradley1, Michael E. Bradley2, James S. Trimmer3, and Burton Horowitz1

1 Department of Physiology and Cell Biology and 2 Pharmacology, University of Nevada, School of Medicine, Reno, Nevada 89557; and 3 Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Voltage-activated K+ (KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the KV currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different KV channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of KV1.4, KV1.5, KV1.6, KV2.2, and KV4.3 transcripts in all regions of the GI tract examined. Transcripts encoding KV1.2, KVbeta 1.1, and KVbeta 1.2 subunits were differentially expressed. KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, KV4.1, KV4.2, and KVbeta 2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these KV channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that KV1.2, KV1.4, KV1.5, and KV2.2 are expressed at the protein level in GI tissues and smooth muscle cells. KV2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of KV channel subunits.

potassium; ion channel; complementary deoxyribonucleic acid; slow wave


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GASTROINTESTINAL (GI) smooth muscles exhibit electrical activity that can vary from slow changes in membrane potential, to fast Ca2+-dependent action potentials. Hyperpolarization and depolarization in membrane potential can also occur in response to neurotransmitters. Rhythmic fluctuations in membrane potential that couple to phasic contractions are termed slow waves (8, 31). Throughout the GI tract, diversity in the electrical activity exists in different organs and in adjacent muscle layers. For example, the colon exhibits slow-wave activity in the circular smooth muscle layer, whereas the longitudinal muscle layer displays a distinctly different electrical activity known as myenteric potential oscillations with superimposed action potentials (13). The electrical properties of the stomach, small bowel, and colon are diverse, differing in resting membrane potential, frequency, and waveform (38). The diversity in electrical activity may be due to the qualitative and/or quantitative expression of ion channels expressed in each region and even within a region of the GI tract.

K+ current contributes to resting membrane potential, duration of rhythmic activity, as well as the response of smooth muscle to neurotransmitters. A number of different K+ conductances have been identified in GI smooth muscles. Both large conductance (BK) (5, 10) and small conductance (23, 49) Ca2+-activated K+ channels have been identified from several species. In addition, inwardly rectifying (4), ATP-sensitive (22, 51) and voltage-dependent Ca2+-insensitive delayed rectifier K+ currents (1, 20, 45, 46) have been recorded from myocytes in several regions of the GI tract.

Delayed rectifier K+ currents (KV) have been shown to play a critical role in the electrical activities of GI smooth muscle. Application of 4-aminopyridine (4-AP), a KV channel blocker, increases slow-wave duration and upstroke depolarization (46). Delayed rectifier K+ current in canine colonic smooth muscle has been shown to consist of three distinguishable components: IdK(f), IdK(s), and IdK(n) (9). IdK(f) is a rapidly activating 4-AP-sensitive current, IdK(s) is a slowly activating tetraethylammonium (TEA)-sensitive current, and IdK(n) is a slowly activating TEA-sensitive current with a low inactivation threshold. Thus delayed rectifier K+ channels form mixed currents that are difficult to fully dissect and characterize individually from native cells. More recently, molecular identification of GI ion channels has contributed to our understanding of the properties of these currents studied in isolation (18). KV channels form the largest and most diverse class of ion channels and can be organized into distinct subfamilies based on the original Drosophila clones: Shaker (KV1), Shab (KV2), Shaw (KV3), and Shal (KV4). Four new subfamilies have been cloned that do not have obvious Drosophila homologues and some have the curious property of not resulting in functional channels when expressed alone [electrically silent channels (37)].

KV channels are composed of tetrameric proteins and may be augmented by the formation of heteromeric channels (21). Although expression of KV alpha -subunits alone can form a functional channel, alpha -subunits have been shown to be associated with auxiliary (beta ) subunits (48). Molecular cloning studies have isolated KVbeta 1.1, KVbeta 1.2, KVbeta 2, and KVbeta 4 genes encoding auxiliary beta -subunits (17, 26, 27, 41). The coexpression of KVbeta -subunits with some KValpha -subunits modulates the channel-gating properties (32) and promotes the surface expression of voltage-gated complexes (42) and hence further enhances the functional diversity of KV channel genes.

In the present study we sought to identify the molecular component underlying KV currents in different regions of GI smooth muscle with the goal of using this information to design genetic knock-out experiments that would effectively correlate expression to function. We show by RT-PCR the differential expression of KV1.2, KV1.4, KV1.5, KV1.6, KV2.2, KV4.3, KVbeta 1.1, and KVbeta 1.2 genes in several GI smooth muscle cells. Expression of KV1.1, KV1.3, KV2.1, KV3.1, KV3.2, KV3.4, and KVbeta 2.1 genes was not detected in any regions of the GI tract examined. For the RNA studies we have used dispersed and isolated GI smooth muscle cells to be certain that the RT-PCR was detecting expression from myocytes and not other heterogeneous cell types within the muscle layer. We also demonstrate that a subset of these channels is expressed at the protein level by immunoblot and immunohistochemical staining of GI tissues.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue dissections and enzymatic isolation of smooth muscle cells. Canine colonic circular and longitudinal smooth muscles were dissected as previously described (7). Canine antrum, duodenum, jejunum, and ileum circular and longitudinal muscles were prepared in a similar manner as the canine colonic smooth muscle tissue. In the tissue preparations, the submucosal and myenteric borders were discarded from the circular and longitudinal muscle preparations. Single smooth muscle cells were dispersed enzymatically as described previously (24).

Isolated-cell preparations. Enzymatically dispersed smooth muscle cells were placed in an experimental chamber under an inverted microscope (Nikon, Japan). The isolated cells were allowed to settle to the bottom of the chamber for ~5 min. A micromanipulator (World Precision Instruments, Sarasota, FL) containing a large-diameter micropipette tip was positioned at the bottom of the chamber in which the cells had attached. Capillary pipettes were made from borosilicate glass capillaries (Sutter Instrument, Navato, CA), pulled on a micropipette puller, and then flame polished to obtain a diameter of ~500 µm. Through applied suction, smooth muscle cells were aspirated into the micropipette. The cells selected were based on the same criteria as those used for electrophysiological examination. Cells that were elongated and spindle shaped (length 200-300 µm, diameter 5-6 µm) were selected. This process was carried out until 60 smooth muscle cells were obtained. Obvious appearing interstitial cells of Cajal were not selected, and c-kit expression was tested for each smooth muscle cell RNA preparation. Some preparations were positive for c-kit expression and were discarded. Cell preparations from at least three different animals were examined to confirm the results. The cells were expunged from the micropipette into an RNase free 0.5-ml tube. The micropipette was washed with Ca2+-free Hanks solution to remove any cells that might have adhered to the glass. The desired cells were snap frozen in liquid nitrogen and stored at -70°C.

Total RNA isolation and RT-PCR. Total RNA was prepared from tissue and isolated smooth muscle cells using SNAP Total RNA isolation kit (Invitrogen, San Diego, CA) as per the manufacturer's instruction including the use of polyinosinic acid (20 µg) as an RNA carrier. Total RNA was also isolated from mouse brain using this method. First-strand cDNA was prepared from the RNA preparations using the Superscript II Reverse Transcriptase kit (GIBCO BRL, Gaithersburg, MD); 500 µg/µl of oligo(dT) primers were used to reverse transcribe the RNA sample. The cDNA reverse transcription product was amplified with channel-specific primers by PCR. PCR was performed in a 50-µl reaction containing 60 mM Tris · HCl (pH 8.5), 1.5 mM MgCl2, 15 mM (NH4)SO4, 10% DMSO, 1 mM dNTP mix, 10 µM of each primer, cDNA, and 1.0 unit of Taq polymerase (Promega, Madison, WI). In a thermal cycler (Coy, Grass Lake, MI) the reaction was denatured at 94°C for 3 min, then followed by 33 cycles (94°C/30 s, 55°C/30 s, 72°C/20 s), and lastly an extension step at 72°C for 10 min. In some reactions step-down PCR was performed. The step-down PCR cycling parameters were denaturation at 94°C for 3 min, followed by 3 cycles (94°C/1 min, 60°C/2 min, 72°C/3 min), 3 cycles (94°C/1 min, 57°C/2 min, 72°C/3 min), 3 cycles (94°C/1 min, 54°C/2 min, 72°C/3 min), 3 cycles (94°C/1 min, 51°C/2 min, 72°C/3 min), 3 cycles (94°C/1 min, 48°C/2 min, 72°C/3 min), 25 cycles (94°C/1 min, 50°C/2 min,72°C/3 min), and a final extension step at 72°C for 7 min. The amplified products (5 µl) were separated by electrophoresis on a 2% agarose-1× TAE (Tris, acetic acid, EDTA) gel, and the DNA bands were visualized by ethidium bromide staining. The PCR products obtained from RNA isolated from the multicell preparation were subjected to a second round of amplification and run on a gel.

Primer design. The following PCR primers were used (the GenBank accession number is given in parentheses for the reference nucleotide sequence used): KV1.1 (L02750) nucleotides (nt) 611-630 and 1301-1320, KV1.2 (L19740) nt 1064-1085 and 1409-1430, KV1.3 (M30441) nt 1045-1064 and 1678-1697, KV1.4 (L02751) nt 1320-1344 and 1943-1962, KV1.5 (U08596) nt 1318-1337 and 1702-1721, KV1.6 (X17622) nt 981-1001 and 1441-1463, KV2.1 (X68302) nt 1439-1458 and 2611-2630, KV2.2 (U69963) nt 184-208 and 985-1007, KV3.1 (Y07521) nt 838-857 and 1560-1581, KV3.2 (M59211) 842-861 and 1538-1558, KV3.4 (M64676) nt 1225-1242 and 1678-1695, KV4.1 (M64226) nt 1630-1649 and 1934-1953, KV4.2 (M59980) nt 330-349 and 862-881, KV4.3 (AF048713) nt 96-112 and 492-509, KVbeta 1.1 (X70662) nt 514-534 and 1127-1147, KVbeta 1.2 (X76723) nt 489-508 and 1034-1053, KVbeta 2.1 (X76724) nt 703-722 and 1239-1258. In addition, PCR primers for beta -actin [(V01217) nt 2383-2402 and 3071-3091] were used to confirm the integrity of the RNA as well as to detect genomic DNA contamination.

Antibodies. The generation and characterization of the anti-KV1.1, KV1.2, KV1.4, KV1.5, KV1.6, and KV2.1 rabbit polyclonal antibodies used in this study were described previously. In brief, antipeptide antibodies were raised that are specific for the KV1.1 (28), KV1.2 (33), KV1.4 (28), KV1.6 (28), and KV2.1 (47) K+ channel alpha -subunit polypeptides. An anti-KV1.5 rabbit polyclonal antibody was raised against a KV1.5 fusion protein (44). A rabbit polyclonal antibody was generated against a glutathione-S-transferase fusion protein, pGEX-KV2.2N, containing amino acids 15-215 of rat KV2.2 (19). This sequence is 96% (194/201) identical to the corresponding sequence of canine KV2.2 (39). In this region rat KV2.2 is very similar to rat KV2.1 (179/201 amino acids, identical = 89%), and thus this polyclonal antibody recognizes both KV2.1 and KV2.2. All rabbit polyclonal antibodies were affinity purified before use.

Mouse monoclonal antibodies against KV1.1, KV1.2, KV1.4, KV1.5, KV1.6, and KV2.1 have been described previously (3). The anti-KV2.2 monoclonal antibody K37/89 (IgG2a) was generated from fusion using a BALB/c mouse immunized with the pGEX-KV2.2N fusion protein. K37/89 was chosen based on its lack of cross-reactivity to KV2.1 by immunofluorescence or immunoblot.

Immunoblotting. The longitudinal and circular smooth muscle layers of the antrum, duodenum, jejunum, ileum, and colon were dissected free of mesenteric fat and connective tissue. Bulk cerebellum was prepared in a similar manner. The tissues were homogenized in sample buffer (50 mM Tris · HCl, 10% SDS, 0.1% bromphenol blue, 10% glycerol, and 5% beta -mercaptoethanol) using a glass-glass Duall homogenizer and then boiled. The crude protein extracts were fractionated on an 8% SDS-PAGE and then electrophoretically transferred to nitrocellulose paper in 25 mM Tris, 192 mM glycine, 20% methanol, 20% SDS buffer. The membranes were blocked overnight with 5% nonfat milk, 0.01% Tween, and Tris-buffered saline (TBS) solution. The blots were incubated with affinity-purified rabbit polyclonal antibodies specific for KV1.1 (1:200 dilution), KV1.2 (1:5,000), KV1.4 (1:5,000), KV1.5 (1:100), or KV2.1 (1:200) channel alpha -subunits for 90 min. The membranes were washed in TBS and 0.04% Nonidet P (NP)-40/TBS for a total of 10 min, incubated in alkaline phosphatase-conjugated secondary antibody (1:7,500 dilution, Promega) for 90 min, washed in TBS, 0.04% NP-40/TBS, and twice with 0.05% Tween/TBS, respectively, for a total of 20 min. The blots were then incubated in substrates for alkaline phosphatase reaction and visualized.

Immunohistochemistry. To examine canine tissues for immunohistochemistry, cryostat sections were employed. Tissues from the proximal colon were opened, and luminal contents was washed with Krebs-Ringer-bicarbonate solution, tissues were pinned onto the base of a Sylgard dish mucosal side up and fixed in 4% paraformaldehyde (wt/vol) made up in 0.01% PBS (0.1 M for 15 min at 4°C). After fixation, tissues were washed for 30 min in PBS (0.1 M, pH 7.4). Tissues were cut into small muscle strips (2 × 10 mm) using a razor blade and cryoprotected in a graded series of sucrose solutions (5, 10, 15, and 20% wt/vol made up in PBS, 1 h each). Tissues were subsequently embedded overnight in a solution containing Tissue Tek (Miles, IL) and 20% sucrose in PBS (1 part/2 parts vol/vol) and rapidly frozen in isopentane precooled in liquid nitrogen. Cryosections were cut on a cryostat at a thickness of 10 µm and collected on Vectabond (Vector Laboratories, Burlingame, CA)-treated slides. Nonspecific antibody binding was reduced by incubation in 10% rabbit serum for 1 h at room temperature. Tissues were incubated overnight at 4°C with monoclonal antibodies (0.1-1 µg/ml) raised against KV channels made up in PBS. Immunoreactivity was detected using FITC-conjugated secondary antibody (FITC-anti-mouse or -rabbit, 1:100 in PBS for 1 h at room temperature). Control tissues were prepared in a similar manner, omitting primary or secondary antibody. Labeled tissues were examined using a conventional Leitz fluorescent microscope or a Bio-Rad MRC 600 confocal microscope with an excitation wavelength appropriate for FITC (496 nm). Confocal micrographs were obtained from digital composites of two-series scans of 10 optical sections through a depth of 10 µm (10 × 1 µm). Final images were constructed with Bio-Rad "Comos" software.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of KValpha - and KVbeta -subunit transcripts in GI smooth muscle cells. To determine the heterogeneity of KV channel subtypes in the GI tract, tissue from antrum, duodenum circular, and the circular and longitudinal muscle layers from the jejunum, ileum, and proximal colon were isolated. With most of the preparations, the regions surrounding the myenteric and submucosal borders were removed due to the prevalence of interstitial cells of Cajal networks expressed in these regions (6, 50).

One goal of our study was to identify the transcriptional expression of K+ channels found in GI myocytes. Circular and longitudinal muscles dissected away from the submucosal and myenteric borders include several cell types, including macrophages, enteric neurons, and interstitial cells of Cajal. Accordingly, we enzymatically isolated smooth muscle cells in the same manner as electrophysiological studies and then selected smooth muscle cells (Fig. 1) for mRNA isolation and RT-PCR analysis. Cells were selected based on the same criteria used to select cells for patch-clamp studies (12, 15). Channel-specific primers were designed from known sequences of KV channel genes to amplify a region specific for each channel isoform. These primers were used in qualitative RT-PCR analysis. The specificity of each amplified PCR product was established by the size of the fragment and confirmed by DNA sequencing. Double amplifications were performed for those experiments in which a single amplification resulted in no product. This assures that low levels of expression for a particular channel would not be missed by the assay. RNA (1 µg) prepared from tissue (either bulk circular or longitudinal muscle with the borders removed) was used to confirm negative results obtained with a set of primers. If a negative result was obtained from tissue-derived RNA it could be assumed that detection level using isolated cells was not a problem and transcript for the channel was indeed not expressed. However, if amplification was positive using bulk tissue as the RNA source yet was consistently negative when using cell-derived RNA it was possible that this channel was either expressed in a minor population of nonmuscle cells or was expressed in smooth muscle cells at an undetectable level for our methods (2 rounds of amplification). KV1.1 was the only channel for which amplification of isolated cells was negative but amplification of bulk tissue-derived RNA was positive.


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Fig. 1.   Phase contrast micrograph of freshly dispersed smooth muscle cells from canine proximal colon circular smooth muscle layer. A: elongated spindle-shaped smooth muscle cell (arrow) that was enzymatically dispersed from canine proximal colon circular smooth muscle tissue. Adjacent to the smooth muscle cell is a micropipette tip. B: application of negative pressure resulted in the smooth muscle cell being aspirated into micropipette (arrow).

In the KV1 family KV1.4, KV1.5, and KV1.6 transcripts were widely expressed in all regions of the GI tract examined (Fig. 2). In contrast, KV1.2 was expressed only in the antrum, ileum circular and longitudinal muscle cells, and colon circular and longitudinal muscle cells. Previous studies using Northern blot analysis demonstrated that KV1.2 is expressed in antrum, duodenum, ileum, and colonic longitudinal and circular smooth muscles (16). Therefore expression in duodenum may be restricted to nonsmooth muscle cells. On the other hand, KV1.1 and KV1.3 transcripts were not detected in GI smooth muscle cells.


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Fig. 2.   Voltage-activated K+ (KV1) channel mRNA expression in gastrointestinal (GI) smooth muscle cells by RT-PCR analysis. mRNA was isolated from smooth muscle cells from several regions of canine GI tract. mRNA was also isolated from positive control brain sample. RT-PCR was performed using gene-specific primers for KV1.1 (A), KV1.2 (B), KV1.3 (C), KV1.4 (D), KV1.5 (E), and KV1.6 (F). Amplified products were separated by electrophoresis on 2% agarose-TAE (Tris, acetic acid, EDTA) gel and then visualized by ethidium bromide staining. Predicted sizes of PCR products were as follows: KV1.1, 709 bp; KV1.2, 374 bp; KV1.3, 652 bp; KV1.4, 641 bp; KV1.5, 404 bp; and KV1.6, 483 bp. A molecular weight marker was also included to illustrate size of the PCR fragments.

In the KV2 family (Fig. 3), the KV2.1 transcript was not found in any region of the canine GI tract examined but was amplified from brain (neuronal) tissue. On the other hand, KV2.2 was distributed throughout the GI tract as well as in the brain. Previous reports have shown that cDNA encoding the KV2.2 channel is ubiquitously expressed in both vascular and visceral smooth muscle preparations (39).


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Fig. 3.   KV2 channel mRNA expression in GI smooth muscle cells by RT-PCR analysis. mRNA was isolated from smooth muscle cells from several regions of canine GI tract. mRNA was also isolated from positive control brain sample. RT-PCR was performed using gene-specific primers for KV2.1 (A) and KV2.2 (B). Amplified products were separated by electrophoresis on a 2% agarose-TAE gel and then visualized by ethidium bromide staining. Predicted sizes of PCR products were as follows: KV2.1, 1,201 bp; and KV2.2, 823 bp. A molecular weight marker was also included to illustrate size of the PCR fragments.

For the KV3 family (Fig. 4), transcripts encoding the KV3.1, KV3.2, and KV3.4 family were identified in the canine brain. Although present in brain, KV3.1, KV3.2, and KV3.4 mRNA could not be detected from myocytes in any region of the canine GI tract examined.


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Fig. 4.   KV3 channel mRNA expression in GI smooth muscle cells by RT-PCR analysis. mRNA was isolated from smooth muscle cells from several regions of canine GI tract. mRNA was also isolated from positive control brain sample. RT-PCR was performed using gene-specific primers for KV3.1 (A), KV3.2 (B), and KV3.4 (C). Amplified products were separated by electrophoresis on a 2% agarose-TAE gel and then visualized by ethidium bromide staining. Predicted sizes of PCR products were as follows: KV3.1, 743 bp; KV3.2, 716 bp; and KV3.4, 470 bp. A molecular weight marker was also included to illustrate size of the PCR fragments.

In the KV4 family (Fig. 5), KV4.1 and KV4.2 channel genes were not identified in the region of the GI tract examined. These transcripts were evident in canine brain. Transcripts for the KV4.3 gene were present in antrum, duodenum circular, jejunum circular, jejunum longitudinal, ileum circular, ileum longitudinal, colon circular, colon longitudinal muscle cells, as well as the brain.


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Fig. 5.   KV4 channel mRNA expression in GI smooth muscle cells by RT-PCR analysis. mRNA was isolated from smooth muscle cells from several regions of canine GI tract. mRNA was also isolated from positive control brain sample. RT-PCR was performed using gene-specific primers for KV4.1 (A), KV4.2 (B), and KV4.3 (C). Amplified products were separated by electrophoresis on a 2% agarose-TAE gel and then visualized by ethidium bromide staining. Predicted sizes of PCR products were as follows: KV4.1, 323 bp; KV4.2, 551 bp; and KV4.3, 413 bp. A molecular weight marker was also included to illustrate size of the PCR fragments.

Examination of the auxiliary KVbeta -subunits (Fig. 6) in canine GI smooth muscle cell preparations detected cDNA encoding the KVbeta 1.1-subunit in jejunum circular, colon circular, and colon longitudinal myocytes. The mRNA expression of KVbeta 1.2 was limited to duodenum circular and jejunum circular myocyte preparations. On the other hand, no KVbeta 2.1 transcript was detectable in any smooth muscle cell preparation.


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Fig. 6.   mRNA expression of accessory KVbeta subunits in GI smooth muscle cells by RT-PCR analysis. mRNA was isolated from smooth muscle cells from several regions of canine GI tract. mRNA was also isolated from positive control brain sample. RT-PCR was performed using gene-specific primers for KVbeta 1.1 (A), KVbeta 1.2 (B), and KVbeta 2.1 (C). Amplified products were separated by electrophoresis on a 2% agarose-TAE gel and then visualized by ethidium bromide staining. Predicted sizes of the PCR products were as follows: KVbeta 1.1, 633 bp; KVbeta 1.2, 564 bp; and KVbeta 2.1, 555 bp. A molecular weight marker was also included to illustrate size of the PCR fragments.

Immunoblot and immunohistochemistry of selected KValpha -subunits in canine GI smooth muscle tissue. Protein expression of KV1.1, KV1.2, KV1.4, KV1.5, and KV2.1 was determined by immunoblot analysis in canine GI smooth muscle (Fig. 7). Crude protein extracts were prepared from antrum, duodenum circular, jejunum circular, ileum circular, and colon circular muscle tissue. Rat brain and canine brain tissue was included with the other preparations as a positive control to show that the polyclonal antibodies were specific for each KV channel polypeptide.


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Fig. 7.   Western blot analysis of selected KV channel subunits in different regions of canine GI tract. Crude protein extracts were prepared from various regions of GI smooth muscle and then fractionated on 8% SDS-PAGE and transferred to nitrocellulose paper. Blots were incubated with appropriate affinity-purified antibody specific for KV1.1 (A), KV1.2 (B), KV1.4 (C), KV1.5 (D), or KV2.1 (E) channel alpha -subunits and then incubated in alkaline phosphatase-conjugated secondary antibody and visualized colorimetrically. Prestained molecular weight marker was also included to ascertain size and mobility of the proteins (not shown). A calculated molecular mass for specific band is included at the left of blot.

In canine brain and GI tissues, the KV1.1 specific polyclonal antibody demonstrated immunoreactivity to an 86-kDa polypeptide, a similar size to that shown in rat brain from previous studies (34). This result is in contrast to the transcriptional analysis in which no amplification of KV1.1 RNA was detected in smooth muscle cells. KV1.1 transcript could be detected from RNA derived from smooth muscle tissue, suggesting that expression may be limited to nonsmooth muscle cells in these preparations. The KV1.2 polyclonal antibody recognized an 88-kDa polypeptide in rat brain, canine brain, as well as all regions of the GI tract examined. The anti-KV1.4 antibody exhibited strong immunoreactivity against an ~96-kDa polypeptide in antrum, duodenum, jejunum, ileum, and colon circular muscles, as well as canine brain. An additional nonspecific band appears to react with the KV1.4 antibody only in the GI samples. Anti-KV1.5 polyclonal antibodies exhibited specific immunoreactivity to a 76-kDa polypeptide present in the membrane fraction of rat brain (44). In rat brain, canine brain, and GI tissue samples a similar 76-kDa protein band was detected by immunoblotting. The KV2.1 specific polyclonal antibody showed specific immunoreactivity to a 100- to 130-kDa polypeptide in rat brain as shown in previous studies (33). The KV2.1 polypeptide was detected in canine brain but was not present in antrum, duodenum, jejunum, ileum, and colon.

Immunohistochemical staining with monoclonal antibodies to KV1.2, KV1.4, KV1.5, KV1.6, KV2.2 and KVbeta 1 (antibody cross-reacts with KVbeta 1.1 and KVbeta 1.2) was performed in canine proximal colon tissue (Fig. 8). Protein expression of KV1.2, KV1.4, KV1.5, KV1.6, KV2.2 and KVbeta 1 was evident in both circular and longitudinal muscle layers of the canine proximal colon. The RNA and protein expression results are summarized in Table 1. Immunoreactivity to the KV channels was not just localized to the plasma membrane of smooth muscle cells but was also observed within the cytoplasm. Other cell types, including blood vessels and enteric neurons, were immunopositive for KV channels. The identities of these cells are currently being studied and will be the focus of future studies.


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Fig. 8.   Immunohistochemistry of KV channel subunits in canine colonic circular and longitudinal smooth muscles. Photomicrographs are digital composites compiled from confocal images of cryostat sections (10 µm). Smooth muscle cells of circular muscle layer (cm) were immunopositive for KV1.2, KV1.4, KV1.5, and KV1.6 (A-D, respectively); note also positive immunoreactivity within blood vessels and submucosal enteric ganglia (eg) in B. Smooth muscle cells of circular and longitudinal muscle (lm) layers were also immunopositive for KV2.2 and KVbeta 1 (E and F, respectively). Where longitudinal muscle is not shown (A-D) positive immunoreactivity was also observed within this muscle layer. Arrows, circular muscularis submucosal interface; arrowheads, septa; asterisks, myenteric plexus between circular and longitudinal muscle layers. Scale bar represents 25 µm in all panels.


                              
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Table 1.   Composite of Kv channel subunit RNA and protein expression in canine GI smooth muscles


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously cloned and characterized KV1.2, KV1.5, and KV2.2 from canine colonic smooth muscle (16, 29, 39). By comparing the properties of these cloned channels to the native delayed rectifier currents we can make tentative assignments of molecular components to these native myocyte K+ currents. KV1.2 and KV1.5 can form heterotetrameric channels with many of the properties of IdK(f) in canine colonic myocytes (36). KV2.2 may be a component of IdK(s) in these same smooth muscle cells (39). With the molecular identification of several colonic KV channels, the question arises as to whether the diverse electrical activity observed in discrete regions of the GI tract may be due to differential expression of ion channels. The objectives of the present study were to comprehensively determine the expression of KV channels in GI smooth muscle cells and to use available antibodies for a subset of KV channels to assess whether transcriptional expression coordinated with protein expression at the smooth muscle tissue level.

The expression of the previously cloned KV1.2, KV1.5, and KV2.2 was confirmed in isolated colonic smooth muscle cells and was found to be expressed in myocytes from other regions of the canine GI tract. A delayed rectifier current with properties similar to those displayed by expression of these cDNAs has been observed in jejunum (14), as well as colon. KV1.4 and KV1.6 transcripts were expressed ubiquitously in myocyte preparations from all regions of the GI tract. Expression of KV1.4 in oocytes results in a delayed rectifier current with rapid inactivation kinetics (30). Native K+ currents from colonic myocytes do not have this property; therefore it is cryptic that KV1.4 is expressed in these cells. However, the expression of KV1.6 in these cells may have a dominant negative effect on inactivation of the native KV1.4 current by forming KV1.4/1.6 heterotetrameric channels and rendering the resulting channels more slowly inactivating (35). Transcripts for KV1.1 and KV1.3 were not detected in any GI smooth muscle cells examined. The absence of KV1.3 from all visceral smooth muscle preparations is not surprising since the distribution of KV1.3 transcripts appears to be limited to thymus, spleen, brain, lung, T-lymphocytes, and pancreatic islet cells (11).

KV1.5 is expressed in canine GI smooth muscle as well as other vascular and visceral smooth muscles (29). This study found that KV1.5 was differentially expressed in the canine GI tract and some longitudinal muscles displayed little or no hybridization using Northern analysis on GI smooth muscle tissue. Using RT-PCR, which is more sensitive than Northern blot analysis, our results indicate that KV1.5 is found in all GI myocytes.

Characterization of the KV3 family in heterologous expression studies has shown that these K+ currents are rapidly inactivating A-type currents, blocked by 4-AP at millimolar concentrations and inhibited by relatively low concentrations of external TEA (40). Presently, no voltage-dependent K+ current that displays these kinetic or pharmacological properties has been described in GI myocytes. In support of those data, cDNAs encoding the KV3.1, KV3.2, and KV3.4 channels were undetectable in all myocyte preparations.

Additional components contributing to the increased functional diversity of KV channels are accessory beta -subunits. Cloning of cDNAs encoding these subunits and expression in heterologous expression systems with specific KValpha -subunits alter the expression level, gating, and conductance properties of the resulting KV channels. KVbeta 1.1 mRNA was detected in jejunum circular, colon circular, and colon longitudinal myocytes, whereas transcripts for KVbeta 1.2 were limited to duodenum circular and jejunum circular myocytes. Coexpression of KVbeta 1.1 with KV1alpha -subunits (with the exception of KV1.6) produces a current that rapidly inactivates. As mentioned previously, no rapidly inactivating A-type current has yet been reported in GI smooth muscle, which may be the result of the ubiquitous expression of KV1.6 in GI myocytes preparations that has a dominant negative effect on N-type inactivation (35).

Although qualitative RT-PCR provides information on the cell specificity of K+ gene expression it does not confirm the presence of an encoded protein. Therefore we performed Western blot analysis and immunohistochemistry to validate that the KV channel mRNA encoded the resultant KV channel protein. Because only a limited number of KV channel antibodies are available, we examined a subset of these KV channels. Immunoblot analysis confirmed that KV1.2, KV1.4, and KV1.5 proteins are present at detectable levels in canine antrum, duodenum, jejunum, ileum, and colon tissue. KV2.1 protein was undetectable in any GI tissue preparation in agreement with results at the mRNA level. Surprisingly, KV1.1 protein was detected in all GI tissues examined, even though KV1.1 mRNA was not evident in any GI smooth muscle cell preparation. Whereas KV1.1 is not expressed in smooth muscle cells it may be found in other cell types (e.g., neurons, interstitial cells of Cajal, macrophages, fibroblasts, endothelial cells, vascular smooth muscle cells, etc). These specialized cell types may contribute significant amounts of protein in smooth muscle tissue crude protein preparations. In addition, KV1.2, KV1.4, KV1.5, KV1.6, and KV2.2 mRNAs are expressed in canine colon circular and longitudinal smooth muscle cells. These results were confirmed by immunohistochemistry of canine colon tissue that showed localization of the KV1.2, KV1.4, KV1.5, KV1.6, and KV2.2 channel proteins to both circular and longitudinal smooth muscle layers.

In conclusion, this study provides a molecular map for the expression of KV-channel subtypes in the canine GI tract at both the mRNA and protein levels. Comparisons between these molecular data, patch-clamp studies at the whole cell level, and sharp electrode recordings of GI electrical activity will allow us to make testable predictions concerning the assignment of molecular components to GI ion channels. The ultimate goal will be to "knock-out" individual channels using molecular approaches, such as murine transgenic models (25) or antisense techniques (2, 43) and determine the effects on GI function and smooth muscle cellular physiology.


    ACKNOWLEDGEMENTS

The National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK-41315 supported this work. J. S. Trimmer is supported by the National Institute of Neurological Disorders and Stroke Grant NS-34383 and is an Established Investigator of the American Heart Association. A. Epperson is a predoctoral fellow of the American Heart Association, Western States Affiliate.


    FOOTNOTES

Present address of K. K. Bradley and M. E. Bradley: Dept. of Pharmacology, Creighton Univ. School of Medicine, 2500 California Plaza, Omaha, NE 68178.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology and Cell Biology, MS 352, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail: burt{at}physio.unr.edu).

Received 15 December 1998; accepted in final form 1 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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