Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy
Takahiro Suzuki and
Koichi Takimoto
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 11 June 2004
; accepted in final form 23 September 2004
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ABSTRACT
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Regulation of voltage-gated K+ (Kv) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating Kv current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitaton followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.
voltage-gated K+ channel; myometrium; gene regulation
THE UTERUS UNDERGOES DRAMATIC CHANGES in size, structure, and contractility during pregnancy. Regulation of membrane excitability plays essential roles in these changes: quiescence of myometrial cells allows expansion of uterus without contraction during pregnancy, whereas synchronous slow-wave contractions are initiated at term. These processes are controlled by fine tuning of Ca2+ manifestation at the plasma membrane via the activity of various ion channels (21, 24). In particular, K+ channels are considered major determinants of excitability and are known to control the membrane potential of various smooth muscle cells (1, 12, 18, 20). Thus changes in the expression and/or function of K+ channels may mediate altered uterus contractility during pregnancy.
Electrophysiological studies have shown that Ca2+-activated and voltage-gated K+ (Kv) currents are present in uterine smooth muscle cell (13, 24). Kv current is further classified into several components with distinct gating properties and sensitivities to drugs. For example, myometrial cells from pregnant humans appear to contain three separable Kv current components (14): 4-aminopyridine (4-AP)-insensitive, 4-AP-sensitive slowly inactivating, and 4-AP-sensitive rapidly inactivating currents. Although the molecular identity of the channels responsible for these currents remains uncertain, several pore-forming and auxiliary subunits are significantly expressed in myometrium. For instance, KvLQT1, human ether-a-go-go-related gene (HERG), and their potential one-transmembrane auxiliary subunits are abundant in these tissues (610, 16). Likewise, significant Kv4.3 mRNA and proteins are found in rat uteri (26). Thus the former and latter channel subunits may constitute the 4-AP-insensitive and the 4-AP-sensitive rapidly inactivating channels, respectively (26).
We wished to further identify molecular components of uterine Kv channels. Specifically, recent studies revealed that functional expression of Kv4 channels requires their association with Kv channel-interacting proteins (KChIPs) (2, 15). Thus the 4-AP-sensitive rapidly inactivating channels in myometrial cells may also contain these auxiliary subunits. To test this possibility, we examined the expression of mRNAs and proteins for KChIPs, as well as Kv4 pore-forming subunits, in rat uterus at various pregnancy stages. Here we report that the expression of Kv4 and KChIP subunits is uniquely and dynamically regulated in rat uterus during pregnancy.
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MATERIALS AND METHODS
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Animals.
The animal investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was performed under the animal protocol approved by the University of Pittsburgh Animal Committee. Sprague-Dawley rats were used at a nonpregnant stage and at various times of pregnancy (gestation days 1315 and 1922) or postparturition (612, 1224, and 2436 h). Whole uterus was taken out and excised into the upper bicornis (corpus) and the lower unicollis (cervix) portions. The obtained uterine tissues were then rinsed with phosphate-buffered saline and immediately frozen on dry ice.
RT and real-time PCR.
RNA was extracted from frozen uterine tissues using the guanidium thiocyanate-based method, following the manufacturer's instruction (RNeasy; Qiagen, Valencia, CA). First-strand cDNA was synthesized with 2.5 µg of total RNA in a 20-µl reaction, using oligo(dT)20 primer and ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA) at 50°C for 1 h. For a standard PCR reaction, 1 µl of the synthesized cDNA was used in a 25-µl PCR reaction containing 2.0 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase (Genechoice, Frederick, MD), and 0.5 µM forward and reverse primers (Table 1). The standard amplification protocol was 2-min denaturation at 94°C and 25 cycles consisting of 94°C for 20 s, 62°C for 20 s, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Different cycle numbers and amounts of cDNAs were used to test the linearity of PCR reactions. The level of mRNA was estimated from the data that were in the linear range with respect to the amount of cDNA used. As negative controls, we used cDNA produced without RT. These negative controls yielded no detectable signals at the expected size in the standard PCR at 35 cycles. PCR products were separated on a 1.8% agarose gel and visualized by ethidium bromide staining. Stained gel images were captured, and band intensities were analyzed using a charge-coupled device camera-based system (BioChem II; UVP, Upland, CA).
Taqman-based real-time PCR for KChIP2 mRNA was performed with primers and probe targeted at a region that is commonly present in all splicing isoforms (29): 5'-GCTGTATCACAAAGGAGGAAATGC-3' (5'-primer, AF269285
[GenBank]
431454), 5'-GAAGCTCTCCACGTGTTCTCTTG-3' (3'-primer, 546524), and 5'-TGACATGATGGGCAAGTACACATACCCTG-3' (probe, 477505). The PCR reaction was done with 200 nM primers and 100 nM 5'-6-FAM/3'-TAMRA-labeled probe (Integrated DNA Technology, Coralville, IA) on an ABI Prizm-7000 machine. A commercially available rat
-glucuronidase kit was used as a normalization control (Applied Biosciences, Foster City, CA). We used the same brain RNA as an internal control for all experiments, and KChIP2 mRNA levels were determined with a threshold cycle (CT) with the comparative CT method using the value obtained with the brain RNA.
Immunoprecipitation and immunoblot analysis.
Anti-KChIP2 sera were raised by injecting hemocyanine conjugated with the first 24-amino acid peptide of rat KChIP2 (MRGQGRKESLSESRDLDGSYDQLTC, in which the last cysteine was introduced for conjugation purposes) in rabbits. Anti-KChIP2 antibody was affinity purified with peptide-linked resin (Sulfolink, Pierce). Anti-Kv4.2 antibodies were obtained from commercial sources (Alomone, Jerusalem, Israel, and Upstate, Chicago, IL). For control experiments, 5 µg each of Myc-tagged Kv4.3 and Emerald [modified green fluorescent protein (GFP)]-tagged KChIP2 or KChIP3 cDNAs were transfected into human embryonic kidney HEK-293 cells on a 100-mm plastic dish by calcium phosphate precipitation, as described previously (29). For immunoprecipitation, affinity-purified anti-KChIP2 antibody was cross-linked to protein G-Sepharose (0.1 ml of resin conjugated with
20 µg of affinity-purified anti-KChIP2 antibody), using a disuccinimidyl suberate-based method (SiezeX; Pierce, Rockford, IL). Immunoprecipitation and immunoblot analyses were performed with the affinity-purified anti-KChIP2 antibody-conjugated resin, according to the previously published method (23).
Uterine tissues were homogenized in 5 vol of 0.25 M sucrose supplemented with 0.2 mM phenylmethylsulfonate, 1 mM iodoacetaminde, and protease inhibitor cocktail (Complete; Roche, Indianapolis, IN), using a polytron-type homogenizer. The postnuclear membrane fraction (P2/3) was obtained by differential centrifugation (27). The pellet membrane fraction was rehomogenized in the Triton-containing lysis buffer [20 mM Tris·HCl (pH 7.5), 0.2 M NaCl, 1 mM EDTA, and 1% Triton X-100] supplemented with protease inhibitors at
5 mg protein/ml, using a Teflon pestle homogenizer. Triton extract was then obtained by centrifugation at 100,000 g for 30 min. Triton extract (
800 µg) was mixed with the antibody-linked protein G-Sepharose (Pierce) for immunoprecipitation.
Immunostaining.
Uterine tissue sections (a middle portion of corpus tissues) were prepared from 4% paraformaldehyde-fixed and paraffin-embedded blocks. Various dilutions of affinity-purified anti-KChIP2 and -Kv4.2 (Alomone) were used for staining transfected cells and sections at 4°C overnight. To confirm the specificity of the KChIP2 signals, preimmune rabbit IgG or affinity-purified antibody preincubated with antigenic peptide was also used in the place of primary antibody. Immunoreactive proteins were detected with indirect fluorescence, using Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes).
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RESULTS
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Expression of Kv4 and KChIP mRNAs is regulated during pregnancy.
We used RT-PCR analysis with primers specific for each Kv4 or KChIP mRNA (Table 1) to estimate their abundances in uterine tissues. All Kv4.14.3 mRNAs were significant in the corpus and cervix of rat uterus at some points during pregnancy (Fig. 1, A and B). Only the long Kv4.3 splicing isoform (28) (GenBank no. U92897) was detected in these tissues. In corpus, the Kv4.3 mRNA level was high in nonpregnant rats and decreased during pregnancy. The observed decrease in Kv4.3 mRNA during pregnancy is consistent with a previous observation (26). In contrast, the Kv4.2 mRNA level increased during pregnancy and declined after parturition. In cervix, the levels of all three Kv4 mRNAs were highest at late gestation. RT-PCR with various amounts of cDNA indicated that Kv4.2 mRNA levels were 2.5 ± 0.7 and3.2 ± 0.6 times larger in corpus and cervix at late pregnancy than in nonpregnant animals [nonpregnant vs. late pregnant, P < 0.05 (2-tailed t-test), n = 3]. Thus the expression of three Kv4 pore-forming subunit mRNAs is uniquely and region selectively regulated in rat uterus during and after pregnancy.

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Fig. 1. Expression of uterine Kv4 mRNA changes during pregnancy. Total RNA was isolated from corpus (A) and cervix (B) of uterus from animals at various pregnant or postparturition periods: nonpregnant (NP), midpregnant (MP, 1315 days in gestation), late pregnant (LP, 1922 days in gestation), 612 h after parturition (PP1), 1224 h after parturition (PP2), and 2436 h after parturition (PP3). RNA from whole brain of adult rats was used as a positive control. RT-PCR was performed with primers specific for indicated voltage-dependent K+ (Kv) channel pore-forming subunits or GAPDH (see Table 1). PCR products were separated on an agarose gel and visualized by staining with ethidium bromide. Sizes of all obtained products corresponded to those expected from their sequences (see Table 1).
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Unlike the significant expression of all three pore-forming subunit genes, we found that KChIP2 and -4, but not KChIP1 or -3, mRNAs were detectable in uterine tissues (Fig. 2, A and B). All three KChIP2 splicing variants (29) were found in both corpus and cervix, with the ratio of the three isoforms being a:b:c =
2:1:4. Two KChIP4 variants (GenBank nos. AAK28291 and AAK28292) were also detectable in rat uterus, with the longer one being predominant. Similar to the pore-forming subunit mRNAs, KChIP2 and -4 message levels were region selectively regulated during and after pregnancy. In both corpus and cervix, KChIP2 mRNAs were very low in nonpregnant animals and markedly increased at late gestation. The KChIP4 mRNA level was significant in nonpregnant animals and slightly reduced during pregnancy in corpus, whereas it exhibited a marked increase at late gestation in cervix. Real-time PCR analysis for KChIP2 mRNAs indicated that the levels of the auxiliary subunit message were 11.4 ± 3.4- and 39.7 ± 17.1-fold higher in corpus and cervix at late gestation than in nonpregnant animals, respectively (nonpregnant vs. pregnant, P < 0.01, n = 3 for each time point; Fig. 2, C and D). Thus the expression of KChIP2 and -4 mRNAs is dynamically and region selectively regulated in uterus during pregnancy.

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Fig. 2. Expression of uterine KChIP mRNAs alters during pregnancy. KChIP mRNAs were detected by RT-PCR analysis with RNAs from corpus (A) and cervix (B) at various pregnant or postparturition periods, as described in the legend for Fig. 1. PCR was performed with primers that distinguish splicing isoforms with different sizes: 3 KChIP2 variants (KChIP2a, -2b, and -2c) (29) and 2 KChIP4 variants (longer and shorter isoforms are presented as 4a and 4b, GenBank nos. AAK28291 and AAK28292). No significant generation of KChIP1 or the 3 products occurred, even after extended PCR (35 cycles). Total KChIP2 mRNA levels were determined by Taqman-based real-time PCR (C and D). Columns and error bars represent means ± SE, respectively (n = 3). *P < 0.01 compared with nonpregnant animals (two-tailed t-test).
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Expression of myometrial Kv4.2-KChIP2 complexes is increased at late gestation.
To test whether the observed changes in Kv4 and KChIP mRNA levels might lead to alterations in their encoding proteins, we examined immunoreactive Kv4.2 and KChIP2 protein levels. We generated an antibody against the first 24-amino acid peptide of the rat KChIP2 protein. This region is present in all three splicing isoforms and shows no apparent similarity to other KChIP gene products (29). When used for standard immunoblot analysis, this antibody gave only a weak signal with uterine KChIP2 proteins (Fig. 3A). Therefore, we used this antibody for immunoprecipitation and subsequent immunoblot analysis to facilitate detection of the auxiliary subunit proteins. Control experiments in a heterologous expression system showed that the affinity-purified anti-KChIP2 antibody effectively immunoprecipitated the target KChIP2 and its associated Kv4.3 proteins (Fig. 3B). Immunoprecipitation, followed by immunoblot analysis, revealed that corpus KChIP2 proteins were significant in uterus at late gestation but were very low in nonpregnant rat tissue (Fig. 3C, middle). Immunoblot analysis with anti-Kv4.2 antibody also showed that significant Kv4.2 proteins are associated with KChIP2 proteins at late pregnancy (Fig. 3B, right). Thus the pregnancy-associated increases in Kv4.2 and KChIP2 mRNAs result in raised expression of complexes containing these subunits.
Various Kv4 pore-forming and KChIP auxiliary subunits may be expressed in distinct cell types. For example, the myometrium contains circular and longitudinal cell layers. Intense Kv4.3 immunoreactivity has been observed in the circular layer of uterus from nonpregnant rats (26). Therefore, the detected Kv4.2-KChIP2 complexes may be selectively expressed in longitudinal cells. It is also possible that these channel complexes are present in nonmyometrial cells. To test these possibilities, we examined cellular localization of Kv4.2- and KChIP2-immunoreactive proteins (Fig. 4). We used a middle portion of corpus tissues for immunostaining. We found that these antibodies detected intense staining in endometrial cells in some, but not all, areas of uterus from late-pregnant rats (Fig. 4A, left). Although the observed regional variation of the detected immunoreactivities might indicate a nonspecific reaction, it is possible that some Kv4.2- and/or KChIP2-immunoreactive proteins are present in these cells. In contrast to the variabilities in endometrium, anti-Kv4.2- or anti-KChIP2-positive cells were consistently found in both circular and longitudinal cell layers of myometrium from late-pregnant rats. In longitudinal cell layers, both immunoreactivities were most obvious in some stretched larger cells, where they were located throughout (Fig. 4B). Unlike uterine tissues of late-pregnant rats, only very weak Kv4.2 or KChIP2 immunoreactivities were detected in uteri of nonpregnant animals (Fig. 4A, right). These localizations suggest that Kv4.2-KChIP2 complexes are present in myometrial cells.

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Fig. 4. KChIP2 and Kv4.2 proteins are expressed in circular and longitudinal myometrial cells at late gestation. A: corpora from nonpregnant (left) and late-pregnant (middle) rats were subjected to indirect immunofluorescent staining with anti-Kv4.2 (top) or affinity-purified anti-KChIP2 (bottom) antibodies. Lower magnifications show entire uterine tissues. Affinity-purified anti-KChIP2 antibody was preincubated with the antigenic peptide (right). EM, LM, and CM, endometrial, longitudinal, and circular muscle layers, respectively. Note that Kv4.2 and KChIP2 immunoreactivities were detected in both longitudinal and circular cells of late-pregnant rats, whereas only weak reactions with these antibodies were seen in tissues from nonpregnant animals. B: higher magnifications illustrate cellular and subcellular localizations in longitudinal cells at late gestation. Note that intense Kv4.2 or KChIP2 immunoreactivities are seen in some stretched larger cells.
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Diffusible factors control the pregnancy-associated regulation of Kv4.2 and KChIP2 mRNA expression.
There are a number of factors that might contribute to the observed pregnant stage-associated regulation of uterine Kv channel gene expression. Notably, sex steroids are known to induce changes in the expression of various proteins, including Ca2+-activated BK (4, 17) and voltage-gated Kv4.3 subunits (26). Female sex steroids are produced mainly by the ovary and distributed to various tissues throughout the body via circulation. Other diffusible factors, such as prostaglandins, act in much shorter distances to influence gene expression within a tissue in which producing cells reside. Finally, mechanical stress induced by developing embryos may also contribute to the altered gene expression. To obtain insight into the regulatory mechanisms, we used uterine tissues of animals that carry embryos in only one side of their bicornis corpora. The one-side conception can be induced by surgical operation; however, it often naturally occurs. Because one-side pregnancy was predicted when animals gave 57 littermates, we obtained pregnant and nonpregnant corpora from these naturally occurring one side-pregnant animals 612 h after parturition. Examining pregnant and nonpregnant corpora in the same animals allows us to test whether pregnant stage-associated changes in diffusible factors, such sex steroids, might be sufficient for inducing altered KChIP2 and/or Kv4.2 expression. RT-PCR analysis revealed that the pregnancy-induced upregulation of Kv4.2 and KChIP2 mRNA expression occurs in both sides (Fig. 5). Using various amounts of cDNAs for PCR and real-time PCR, we estimated that Kv4.2 and KChIP2 mRNA levels in the pregnant side were 121 ± 37 and 124 ± 67% of those in the nonpregnant side, respectively (n = 3). These data suggest that circulating hormonal factors largely account for the pregnancy-associated increases in Kv4.2 and KChIP2 mRNA expression.

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Fig. 5. KChIP2 and Kv4.2 mRNA levels are similarly increased in pregnant and nonpregnant corpora of one-side-conceived rats. RT-PCR analysis was performed with total RNA isolated from corpora of rats that carried embryos on one side at 612 h after parturition and nonpregnant animals. Representative RT-PCR results for Kv4.2 and KChIP2 are shown. Kv4.2 mRNA level in the pregnant side was estimated to be 121 ± 37% of that in the nonpregnant side (n = 3). Real-time PCR analysis indicated that the KChIP2 mRNA level in the pregnant side was 124 ± 67% of the nonpregnant side.
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DISCUSSION
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The rapidly inactivating 4-AP-sensitive K+ current is prominent in myometrial cells (11, 19, 22, 25, 30, 31) and plays an important role in controlling membrane potential during gestation (19, 25). The kinetics and pharmacological properties of this current resemble those produced by channel subunits in the Kv4 family. However, recent studies revealed that native Kv4 channels are composed of pore-forming and KChIP auxiliary subunits (2, 15). Consistent with this notion, the expression of KChIPs is found in smooth muscle cells from various tissues (1). In this study, we examined the expression and cellular localization of Kv4 pore-forming and KChIP auxiliary subunits. Our data indicate that all three pore-forming (Kv4.14.3) and two auxiliary (KChIP2 and -4) subunits are significantly expressed in rat uterus. Furthermore, the expression of these subunits is dramatically regulated during and after pregnancy. Specifically, Kv4.2 and KChIP2 mRNAs and proteins are markedly increased in late gestation. Immunostaining also showed that these two proteins are similarly distributed in circular and longitudinal myometrial cells at late pregnancy. These results suggest that Kv4.2-KChIP2 complexes constitute a significant portion of the rapidly inactivating current in myometrial cells at late gestation. They also indicate that molecular compositions of the rapidly inactivating channels in myometrial cells change during pregnancy.
Electrophysiological studies revealed that myometrial cells contain at least three Kv current components that differ in gating properties and sensitivities to drugs. In addition to the rapidly inactivating current, 4-AP-sensitive and -insensitive slow-inactivating components are seen in these cells. Because channels encoded by Kv14 families are sensitive to 4-AP, the drug-sensitive slow-inactivating currents are likely generated by these channel subunits. For example, we found abundant expression of some Kv1 family, Kv2.1, and silent Kv9.3 subunit mRNAs in rat uterus (unpublished observation). Thus Kv1.x and/or Kv2.1-Kv9.3 complexes may significantly contribute to the 4-AP-sensitive slow-inactivating component. Furthermore, it has been shown that mRNAs for KvLQT1 (KCNQ1) and HERG (KCNH1), as well as their potential one-transmembrane auxiliary subunits (KCNEs) (510, 16, 22), are highly expressed in the uterus. Therefore, the 4-AP-insensitive component with rather unusual gating properties may be carried by KCNQ- and/or KCNH-KCNE channel complexes.
Sex steroids have been shown to regulate expression and splicing of various ion channels in uterus. For example, estrogens appeared to alter the expression and subcellular localization of Kv4.3 proteins (26). Likewise, the expression levels of Ca2+-activated BK channel pore-forming
- and auxiliary
-subunits are altered by pregnancy and estrogens (4, 17). In addition, these hormones were found to induce the switch in splicing pattern of the
-subunit (3). Finally, mRNA and protein levels for the one-transmembrane K+ channel auxiliary subunit, minK, are dramatically changed during pregnancy and by estrogens (5, 6, 8, 22). Our experiments with one side-pregnant animals indicate that the pregnancy-associated upregulation of Kv4.2 and KChIP2 mRNAs is largely accounted for by diffusible factors. Hence, diffusible hormonal factors, such as sex steroids, may play important roles in orchestrating the expression and splicing of various K+ channel pore-forming and auxiliary subunits. However, changes in estrogen levels may not be sufficient to induce the observed dramatic changes in Kv4.2 and KChIP2 gene expression. Although we did not measure estrogen levels in nonpregnant animals, Kv4.2 or KChIP mRNA levels were relatively stable in all nonpregnant animals. This suggests that the estrous cycle-related changes in hormone levels are not sufficient to produce dramatic regulation of channel subunit gene expression. Rather, sustained elevations in these sex steroids or additional factors, the levels of which alter during pregnancy, may be required for the induction of the pregnancy-associated changes in Kv4.2 and KChIP2 gene expression.
A previous study has shown that Kv4.3 expression is reduced during pregnancy. We also observed a similar reduction in the Kv4.3 mRNA level. In addition, the KChIP4 mRNA level appeared to decrease during pregnancy in corpus of rat uterus. Therefore, it is tempting to speculate that Kv4.3-KChIP4 channel complexes are major forms of the rapidly inactivating channels in nonpregnant animals, whereas they are replaced with Kv4.2-KChIP2 complexes during pregnancy. This potential subunit composition switch may occur in the same cells or distinct cell populations. While it has been shown that Kv4.3 proteins are mostly present in circular myometrial cells, our immunostaining revealed that Kv4.2 and KChIP2 proteins are present in both circular and longitudinal cells. Thus the observed changes in subunit compositions occur at least in part in distinct cell populations. Further studies with cell isolation from individual layers, as well as the use of toxins specific for each Kv channel type, may be required to unambiguously resolve these issues.
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ACKNOWLEDGMENTS
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Present address of T. Suzuki: Dept. of Obstetrics and Gynecology, Sapporo Medical University, Sapporo, Hokkaido 060-8543, Japan.
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FOOTNOTES
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Address for reprint requests and other correspondence: K. Takimoto, Dept. of Environmental and Occupational Health, Univ. of Pittsburgh, 3343 Forbes Ave., Pittsburgh, PA 15260 (E-mail: koichi{at}pitt.edu)
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. Section 1734 solely to indicate this fact.
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