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
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ABSTRACT |
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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,
KV1.1, and
KV
1.2 subunits were
differentially expressed. KV1.1,
KV1.3,
KV2.1,
KV3.1,
KV3.2,
KV3.4,
KV4.1,
KV4.2, and
KV
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
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INTRODUCTION |
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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 -subunits alone can form a
functional channel,
-subunits have been shown to be associated with
auxiliary (
) subunits (48). Molecular cloning studies have isolated
KV
1.1, KV
1.2,
KV
2, and
KV
4 genes encoding auxiliary
-subunits (17, 26, 27, 41). The coexpression of
KV
-subunits with some KV
-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,
KV1.1, and
KV
1.2 genes in several GI
smooth muscle cells. Expression of
KV1.1,
KV1.3, KV2.1,
KV3.1,
KV3.2,
KV3.4, and
KV
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.
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MATERIALS AND METHODS |
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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, KV1.1 (X70662) nt 514-534 and 1127-1147,
KV
1.2 (X76723) nt 489-508 and 1034-1053, KV
2.1
(X76724) nt 703-722 and 1239-1258. In addition, PCR primers
for
-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 -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.
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% -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
-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.
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RESULTS |
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Identification of KV- and
KV
-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).
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Immunoblot and immunohistochemistry of selected
KV-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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DISCUSSION |
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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 -subunits. Cloning of cDNAs encoding these subunits and
expression in heterologous expression systems with specific
KV
-subunits alter the
expression level, gating, and conductance properties of the resulting
KV channels.
KV
1.1 mRNA was detected in
jejunum circular, colon circular, and colon longitudinal myocytes,
whereas transcripts for KV
1.2
were limited to duodenum circular and jejunum circular myocytes.
Coexpression of KV
1.1 with
KV1
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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