Protein distribution of Kcnq1, Kcnh2, and Kcne3 potassium channel subunits during mouse embryonic development
Article first published online: 6 FEB 2006
Copyright © 2006 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 288A, Issue 3, pages 304–315, March 2006
How to Cite
de Castro, M. P., Aránega, A. and Franco, D. (2006), Protein distribution of Kcnq1, Kcnh2, and Kcne3 potassium channel subunits during mouse embryonic development. Anat. Rec., 288A: 304–315. doi: 10.1002/ar.a.20312
- Issue published online: 20 FEB 2006
- Article first published online: 6 FEB 2006
- Manuscript Accepted: 5 DEC 2005
- Manuscript Received: 9 AUG 2005
- Ministry of Science and Technology, Spain. Grant Number: SAF2002-3436-C02-01
- Junta de Andalucia Regional Council, Spain. Grant Number: CTS446
- ion channel;
Voltage-dependent potassium channels consist of a pore-forming α-subunit, which is modulated by additional β-ancillary or regulatory subunits. Kcnq1 and Kcnh2 α-channel subunits play pivotal roles in the developing and adult heart. However, Kcnq1 and Kcnh2 have a much wider expression profile than strictly confined to the myocardium, similar to their putative regulatory Kcne1-5 β-subunits. At present, the distribution of distinct potassium channel subunits has been partially mapped in adult tissues, whereas almost no information is available during embryonic development. In this study, we report a detailed analysis of Kcnq1, Kcnh2, and Kcne3 protein expression during mouse embryogenesis. Our results demonstrate that Kcnq1 and Kcnh2 are widely distributed. Coexpression of both α-subunits is observed in a wide variety of organs, such as heart and the skeletal muscle, whereas others display unique Kcnq1 or Knch2 expression. Interestingly, Kcne3 expression is also widely observed in distinct tissue layers during embryogenesis, supporting the notion that an exquisite balance of α- and β-subunit expression is required for modulating potassium conductance in distinct organs and tissue layers. © 2006 Wiley-Liss, Inc.
Ion channels play important roles in both excitable and nonexcitable cells (Hatta et al.,2002). Voltage-dependent sodium, calcium, and potassium channels have been involved in a wide variety of cell functions (Snyders,1999). In particular, potassium channels have been reported to play critical roles in the repolarization phase of the cardiac action potential as well as in the secretory release of several components in the digestive tract epithelial layer (Melman et al.,2001).
Potassium channels belong to a complex protein superfamily, encoded by a large number of genes (Pongs,1999; Pongs et al.,1999). A single potassium channel gene encodes a unique subunit, which can participate on the formation of several functionally distinct homomeric or heteromeric channels (Polvani et al.,2003). Voltage-dependent ion channels consist of a pore-forming component, the α-subunit, and additional ancillary or regulatory β-subunits. The voltage-dependent potassium channel α-subunits contain two to six transmembrane regions and, in most cases, four subunits are required for the assembly and formation of functional potassium channel (Hatta et al.,2002). In native cells, potassium channels incorporate additional subunits, ancillary or regulatory β-subunits, that modify attributes such as surface half-life, ion selectivity, gating kinetics, chemical and secondary messenger regulation, and pharmacological properties (Abbott and Goldstein,2001).
Potassium channels are widely distributed within distinct cell and tissue layers in the adult mouse, playing pivotal roles in cell signaling and electrophysiological events (Sands et al.,2005). Mutations in several components of the voltage-dependent potassium channel family such as Kcnq1 and Kcnh2, as well as in the Kcne family of β-subunits, have been related to arrythmogenic syndromes such as long QT (Abbot and Goldstein,2002). Kcnq1 is alternatively spliced, yielding a full-length isoform and a novel truncated variation termed kcnq1b, which have a 63 amino acid truncation at the C-terminus. Kcnq1b was not detected in heart or brain but represented approximately half the kcnq1 transcripts expressed in murine portal vein (Ohya et al.,2003). In mammalian heart, two Kcnh2 transcripts (ERG1a and 1b) encode proteins differing in their amino-terminal sequence and gating properties. There are evidences for ERG1b expression, localization, and coassembly with ERG1a in cardiac ventricular myocytes. These findings indicate cardiac IKr channels are composed of both ERG1a and ERG1b α-subunits (Jones et al.,2004).
Curiously, Kcnq1 and Kcnh2 have a much wider expression profile than strictly the myocardium (Demolombe et al.,2001). Kcnq1 is prominently expressed in stomach, small intestine, colon, kidney, heart, inner ear, and in airway cells in the adult stage, whereas Kcnh2 is expressed in testis (London et al.,1997), thymus, neuronal tissues, heart, and retina (Polvani et al.,2003). Interestingly, heterologous expression studies in Xenopus oocytes have described that coexpression of Kcnq1 and the Knce1 ancillary subunit produces slowly activating potassium currents that resemble cardiac IKs, whereas coexpression of the Knch2 pore-forming subunit and the Kcne2 ancillary subunit produces similar currents to the rapidly activating cardiac IKr (Nerbonne,2000). These observations led to the paradigm that association of Kcnq1 + Kcne1 and Kcnh2 + Kcne2 governs the cardiac IKs and IKr repolarization currents, respectively (Nerbonne et al.,2001).
In the human heart, the delayed rectifier rapid (IKr) and slow (IKs) currents are generated by voltage-gated potassium channels. IKr is characterized by rapid activation of −30 mV, rapid inactivation, and strong inward rectification at positive potentials, which is due to rapid voltage-dependent C-type inactivation. When ERG is coexpressed with Kcne2, it shifts the ERG activation current in the positive direction, accelerating single-channel conductance (Tamargo et al.,2004). IKs is slowly activated at potentials positive to −30 mV with a linear current of 20 mV. Thus, IKs contributes to human atrial and ventricular repolarization, particularly during action potentials of long duration and is a dominant determinant of the physiological heart rate-dependent shortening of cardiac action potential duration (APD).
However, three novel Kcne (Kcne3, Kcne4, and Kcne5) family members have been recently reported, which are also widely distributed in the adult mouse (Grunnet et al.,2002,2003; Teng et al.,2003). Coexpression experiments of Kcnq1 and Kcne3 subunits give rise to a constitutively opened pore (Mazhari et al.,2002), whereas coexpression with either Kcne4 or Kcne5 suppresses the current IKs (Lundquist et al.,2005). Thus, a critical balance of distinct β-subunit distribution in relationship with the potassium channel α-subunits seems to be compulsory.
At present, potassium channel subunit distribution has been partially mapped in some adult tissues, whereas very little information is available on the expression and putative functional role of voltage-dependent potassium channels during embryogenesis. We previously described the transcript expression profiles of Kcnq1, Kcnh2, Kcne1, Kcne2, and Kcne3 in the developing heart (Franco et al.,2001b). Our observations suggested a critical role of β-subunits in the chamber-specific cardiac repolarization based on their differential gene expression profile in the developing heart.
In this article, we have mapped the protein expression profile of Kcnq1 and Kcnh2 potassium channel α-subunits versus Kcne3 because of its specific modulation of α-subunits gating. Our results demonstrate that Kcnq1 and Kcnh2 are widely distributed also during embryonic development. Coexpression of both pore-forming subunits is observed in a wide variety of organ primordia, such as the heart and the skeletal muscle, whereas other display unique expression of either Kcnq1 or Knch2. Interestingly, Kcne3 expression is also widely observed in distinct tissue layers during embryogenesis. Its putative role in relationship with Kcnq1 and Kcnh2 is discussed.
MATERIALS AND METHODS
BALB/c mouse embryos were isolated ranging from embryonic day (E) 8.5 to E18.5. Mice were handled and sacrificed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The tissues were washed in PBS and fixed overnight in 4% paraformaldehyde. Subsequently, they were dehydrated, embedded in paraplast, and 15 μm tissue sections were cut and mounted into poli-L-lysine-treated slides as previously described (Franco et al.,2001a).
Tissue samples were deparaffinated, rehydrated in decreasing graded ethanol steps, and briefly rinsed in PBS. Nonspecific binding was minimized by TENG-T incubation for 15 min. Subsequently, the sections were incubated overnight with the corresponding primary antibody. The polyclonal antibodies used in these experiments were goat anti-Kcnq1 (K-17; sc-10645; Santa Cruz, CA), rabbit anti-Kcnh2 (APC-062; Alomone Labs, Tel Aviv, Israel), and goat anti-Kcne3 (N-18; sc-10647; Santa Cruz).
The anti-kcnq1 and anti-kcnh2 antibodies recognized both spliced variants of Kcnq1 and Kcnh2 genes, respectively. The monoclonal antibody anti-smooth muscle actin (A5228; Sigma) was used to delineate the smooth muscle components. Primary antibodies were removed by a brief rinse in PBS and subsequently the sections were incubated with the corresponding secondary antibody conjugated with alkaline phosphatase (Sigma) for 90 min. Thereafter, the sections were washed in PBS and coloration was revealed by NBT/BCIP solution for 20 min. Finally, the samples were dehydrated and mounted in DPX (Franco et al.,2001a). The specificity of the primary antibody was assessed by lack of primary antibody incubation, which resulted in all cases in no detectable staining (data not shown).
We have analyzed the expression profile of the pore-forming Kcnq1 and Kcnh2 potassium channel subunits and the Kcne3 ancillary subunit during mouse embryonic development. The expression of Kcnq1, Kcnh2, and Kcne3 is described only in those tissue layers and organs in which expression was observed (Table 1).
|Kidney||external fibrous capsule||+/−||+||+/−|
|Bladder||external longitudinal layer||+||+/−||+|
|Esophagus||smooth muscle layer||++||+||+|
|inner epithelial lining||+||+||+|
|Stomach||smooth muscle layer||++||+/−||+|
|inner epithelial lining||+||+/−||+|
|Gut||smooth muscle layer||+||+/−||+|
|NERVOUS & SENSORY ORGANS|
|Spinal cord||neural tube||+||+||+|
|dorsal root ganglia||+||++||+|
Expression Profile in Urinary System
The expression of the Kcnq1, Kcnh2, and Kcne3 potassium channel subunits can be observed for the first time at E11.5 in the mesonephric duct (Fig. 1A–C). With further development, expression of each potassium channel subunits diverged within the developing kidney.
Kcnh2 and, to a lesser extent, Kcnq1 and Kcne3 are expressed in the external fibrous capsule of the kidney (Fig. 1D–F). On the other hand, Kcnq1 and Kcne3 are homogeneously expressed along the convoluted and collecting tubules, whereas Kcnh2 is only expressed in the collecting tubules (Fig. 1D–F). Kcnq1 and Kcne3 are also highly expressed in the urether, whereas no expression of Kcnh2 can be observed. No expression of Kcnq1, Kcnh2, and Kcne3 is observed in the mesenchymal tissue.
The bladder is basically formed by three muscular layers, an external longitudinal, a mid-circular, and an internal layer covered internally by a transitional epithelial layer. At E14.5, Kcnq1 and Kcne3 expression can be observed in all muscle layers as well as in the internal epithelial lining. Curiously, only a weak Kcnh2 expression can be observed in the muscular layers of the bladder (Fig. 1G–I).
The first expression of Kcnq1, Kcnh2, and Kcne3 in the urethra is seen at E14.5. The external epithelium, which surrounds the urethra, showed expression of Kcnq1, Kcnh2, and Kcne3, whereas the internal epithelium displayed only Kcne3 expression (Fig. 1J–L). These expression patterns are maintained during further embryonic development.
Expression Profile in Digestive System
The expression in the esophagus can be observed for the first time at E11.5, when Kcnq1 expression can be documented in the surrounding smooth muscle layer (Fig. 2A–C). Kcnh2 and Kcne3 potassium channel subunits are expressed for the first time at E14.5, with an identical expression profile as that previously described for Kcnq1 (Fig. 2G–I). Kcnq1 expression was more intense as compared to Kcnh2 expression in the smooth muscle layer. In addition, expression of Kcnq1, Kcnh2, and Kcne3 can also be observed in the inner epithelial lining of the esophagus at this stage (Fig. 2G–I). Such expression profile is maintained with further embryonic development. α-SMA was used to delimit the smooth muscle component within the esophagus as shown in Figure 2J.
The first expression in the primitive stomach (hind-gut) can be observed in stage E10.5 only for the potassium subunits Kcnq1 and Kcne3. There was no expression of the α-subunit Kcnh2 at this stage (Fig. 2D–F). With further development, at E14.5, Kcnq1, Kcnh2, and Kcne3 expression can be observed in distinct tissue layers of the developing stomach. Kcnq1 and Kcne3 are widely expressed in the smooth muscle layer, the connective tissue layer, and the internal epithelial lining. Curiously, the expression levels were lower in the connective tissue as compared to the smooth muscle layer. Furthermore, Kcnq1 expression was high as compared to Kcne3 (Fig. 2K–M). Kcnh2 is weakly expressed in all the stomach tissue layers as compared to Kcnq1 and Kcne3 expression. Kcnh2 is moderately expressed in the inner epithelium and only basal levels were detectable in the smooth muscle layer (Fig. 2K–M). In the stomach, the smooth muscle layers were delimited by α-SMA immunostaining (Fig. 2N).
Kcnq1 and Kcne3 expression is first observed at E10.5, displaying a similar pattern along the anteroposterior axis in the developing gut (Fig. 2D–F). Both potassium channel subunits are expressed in the smooth muscle layers of the intestine (Fig. 2O and Q). Kcne3 also displayed basal expression levels in the epithelial layer, whereas no detectable expression of Kcnq1 can be observed in the intestinal epithelium. Kcnh2 is weakly expressed in the smooth muscle layer but intensively expressed in the epithelial lining from E12.5 onward (Fig. 2P). α-SMA immunostaining displays the location of the smooth muscle layers in the gut (Fig. 2R).
The first expression of potassium channel subunits in the liver is documented at E14.5 and exclusively for Kcnh2 (Fig. 2T), basically confined to the developing hepatocytes. There was no expression of Kcnq1 and Kcne3 during liver development (Fig. 2S and U). Expression of α-SMA was basically not detectable in the liver (Fig. 2V).
Expression Profile in Cardiovascular System
The expression pattern differs between the pore-forming subunits, Kcnq1 and Kcnh2, and the auxiliary β-subunit Kcne3 during cardiac embryonic development. Kcnq1 and Kcnh2 are homogeneously expressed within the atrial and ventricular myocardial chambers during development, ranging from E9.5 to birth (Fig. 3A, C). However, the expression pattern of Kcne3 was more heterogeneous. At early stages, E9.5, Kcne3 expression is observed along the entire myocardium (Fig. 3C). From E16.5 onward, Kcne3 expression became regionalized (Fig. 3F), being higher expressed in the atrial chamber as compared with the ventricular chamber myocardium. Such atrial confined expression remains similar in later fetal stages of development.
Kcnq1, Kcnh2, and Kcne3 expression is observed in the forming blood vessel endothelium at early stages of development (Fig. 3G–I). With further development, Kcnh2 and Kcne3 displayed a similar expression profile within the arterial and venous blood vessels, which is basically confined to the endothelial lining (Fig. 3J–L). However, a differential Kcnq1 protein expression profile is observed between arterial and venous components. Kcnq1 is expressed in both endothelial and smooth muscle layers in the arterial blood vessels (Fig. 3J), but no detectable expression is observed in these layers on the venous blood vessels.
Expression Profile in Respiratory System
At E10.5, robust Kcnh2 and moderate Kcnq1 expression is observed in the internal epithelial lining of the trachea, whereas Kcne3 was basically undetectable (Fig. 2A–C). During further development, the Kcnq1 and Kcnh2 remain to be expressed at similar levels in the tracheal epithelium, whereas Kcne3 displayed a moderate expression at E14.5 (Fig. 4D–F).
Kcnq1, Kcnh2, and Kcne3 expression is first observed at E10.5 within the primitive lung buds, confined to the epithelial lining, being more robust for Kcnh2 as compared to Kcnq1 and Kcne3 (Fig. 4A–C). With further development, the expression of Kcnq1, Kcnh2, and Kcne3 can be observed in the epithelial lining of the bronchi and bronchioles, whereas no expression can be observed in the alveolar epithelia (E14.5; Fig. 4G–I). Expression of Kcnq1 ancillary subunit was weaker as compared to Kcnh2 and Kcne3 (Fig. 4J–L). Such expression profile remains unchanged during further lung development.
Expression Profile in Nervous System and Sensorial Organs
The architecture of the nervous system is highly dynamic and complex, deserving therefore a detailed and independent study. In this article, we only describe the expression profile of Kcnq1, Kcnh2, and Kcne3 in the spinal cord as well as in the most prominent sensorial organs.
The expression of these potassium channel subunits is observed from the first stages of the embryonic development in E10.5 (Fig. 5A–C). From this stage onward, Kcnq1, Kcnh2, and Kcne3 are expressed in the neural tube and subsequently in the dorsal root ganglia (Fig. 5D–F). The expression of α-SMA was basically not detectable in the neural tube (Fig. 5G).
The vibrissae are sensory organs located at the upper lip of the bucal cavity, consisting basically on hair follicle structures. Kcnq1, Kcnh2, and Kcne3 displayed strong expression in the epithelial layer of the vibrissae primordia (Fig. 5H–J) during fetal development. α-SMA expression was only faintly observed in the smooth muscle cells around the vibrissae epithelial layer (Fig. 5K).
Inner ear (cochlea).
We have observed only a weak Kcnh2 and Kcne3 expression exclusively confined to the cochlear epithelial lining, starting at E14.5, whereas no expression of Kcnq1 has been observed during embryonic and fetal development (Fig. 5L–N). There was basically no detectable expression of α-SMA in the inner ear during the different developmental stages analyzed (Fig. 5O).
The eye is a complex structure derived from optic placode. During development, several key structures are progressively established, such as the crystalline, the iris, as well as specific tissue layers such as the cornea and the retina. Expression of Kcnq1, Kcnh2, and Kcne3 was not observed at early developmental stages (data not shown). First expression is observed for Kcnq1 and Kcne3 at E14.5, confined to the crystalline lens and the retina (Fig. 5P and R). Curiously, Kcnh2 expression was absent in the different structures during eye development (Fig. 5Q). In the developing eye, the expression of α-SMA was only detected in the smooth muscle component around the retina and the optic nerve (Fig. 5S).
Expression Profile in Skeletal Muscle
Kcnq1, Kcnh2, and Kcne3 potassium channel subunits are expressed in skeletal muscle during embryogenesis (Fig. 6), including cranial, thoracic, and limb regions.
Functional diversity of potassium channel activity in mammalian tissues is widely documented (Sands et al.,2005). Mutations in several components of the voltage-dependent potassium channel family such as Kcnq1 and Kcnh2 have been related to arrythmogenic syndromes such as long QT (Abbott and Goldstein,2002). Curiously, Kcnq1 and Kcnh2 have a much wider expression profile than merely within the myocardium (Demolombe et al.,2001). Kcnq1 and Kcnh2 have been reported to be expressed in distinct adult organs by RNAse protection assays and RT-PCR techniques (London et al.,1997; Demolombe et al.,2001; Polvani et al.,2003). However, to date, a single study has described the protein expression profile of these potassium channel subunits at the tissue level (Franco et al.,2001b), whereas very little information is available on their onset of protein expression and tissue distribution during embryogenesis. In this study, we provide a comprehensive overview of the protein expression profile of Kcnq1 and Kcnh2 during embryogenesis.
We have observed that Kcnq1 and Kcnh2 proteins are expressed in a wide variety of organs during embryogenesis in line with previous data in adult stages (Demolombe et al.,2001). Several tissues such as the developing smooth, skeletal, and cardiac muscle display coexpression of Kcnq1 and Kcnh2 (Table 1). Curiously, there are several cell tissue layers that exclusively display expression of either Kcnq1 or Kcnh2 (Table 1). These data support the hypothesis that Kcnq1 and Kcnh2 have distinct roles that might be also different within distinct cell tissue layers. An independent discussion within the distinct developmental systems studied in this work is provided below.
K Channel Pore-Forming Subunits in Developing Urinary System
In the adult kidney, Kcnq1 protein has been detected in the luminal membrane of proximal tubular cells (Choaube et al.,1997; Wang,2004), attributing therefore a putative role in the nephron (Bleich and Warth,2000; Wang,2004). We have detected Kcnq1 and Kcnh2 protein expression in the collecting tubules, whereas only Kcnq1 protein was observed in the convoluted tubules and the urether of the developing kidney. These data suggest an early potassium regulation in the developing kidney, which seems to be differential between convoluted and collecting tubes. Such potassium regulation seems also to be effective in the bladder and urethra epithelia. Furthermore, we have observed expression of Kcnq1, Knch2, and Knce3 in the surrounding fibrous capsule. To date, the putative role of these proteins in this tissue remains elusive.
K Channel Pore-Forming Subunits in Developing Digestive System
We have observed several patterns of expression of the Kcnq1 and Kcnh2 potassium channel subunits during the development of the digestive tract. Kcnq1 and Kcnh2 are coexpressed along the epithelial and smooth muscle layers of the digestive tract from early stages of development, spanning from the esophagus until the stomach. Previous studies in the adult digestive tract have provided evidence of Kcnq1 expression in the parietal cells of the stomach (Dedek and Waldegger,2001) as well as in crypt cells of the colon and small intestine (Bleich and Warth,2000). A putative role in potassium conductance-mediated acid and/or chloride secretion of Kcnq1 in these cells have been proposed (Liao et al.,2005). Our data support that potassium conductance in the epithelial lining of the digestive tract seems to be established at an early stage of development. An alternative role of Kcnh2 expression in the developing digestive tract, at least in the embryonic gut and liver, where expression is only observed during embryogenesis, might be contributing to control cell proliferation, as it has been suggested in other tissues (Pardo,2004). Furthermore, we have also observed that Kcnq1 and Kcnh2 are widely expressed in the developing smooth muscle layers of the digestive tract, supporting the notion that contraction of the smooth muscle cells can be elicited at such early stage, contributing in some degree to the peristatoid contraction of the digestive tract.
Curiously, expression within the developing intestine is rather peculiar since Kcnq1 is not observed in the epithelial lining, but Kcnh2 is highly expressed, whereas Kcnh2 is no longer observed in the smooth muscle lining, but high expression of Kcnq1 is documented. These observations suggest that specific potassium conductance-mediated contraction is unique for the intestinal tract at least at early stages of development. In the same line, Kcnh2 seems to be the prominent pore-forming subunit expressed in the developing gut, whereas in the adult intestinal epithelia Kcnq1 is widely documented (Delomombe et al.,2001; Warth et al.,2002). This discrepancy might be due to progressive shift in the potassium modulation requirements of the developing intestinal epithelial as maturation processes. In this line of thinking, a progressive replacement of Kcnh2 would take place in favor of Kcnq1. Such a shift might take place at neonatal stages, when the functional intestine maturation is acutely occurring (Uhlig et al.,2004).
K Channel Pore-Forming Subunits in Developing Cardiovascular System
We have previously documented that Kcnq1 and Kcnh2 are homogeneously distributed in the developing heart from early stages of development (Franco et al.,2001b), in line with the results obtained in this study. The role of these potassium channels in the cardiac repolarization phase of the action potential is widely acknowledged within the developing (Davies et al.,1996) and adult myocardium (Charpentier et al.,2004).
Kcnq1 and Kcnh2 display a differential expression profile between different tissue layers in the arterial and venous blood vessels. Kcnq1 is expressed in the endothelial and smooth muscle layers of the arterial but not of the venous blood vessels. On the other hand, Kcnh2 is expressed in the endothelial but not in the smooth muscle layer of both arterial and venous blood vessels during development. This is the first report that illustrates expression of Kcnq1 and Kcnh2 in the endothelial blood vessel layers. Their role remains to be determined, although it seems that it is clearly established at early stages of vascular development.
In the adult stage, both Kcnq1 and Kcnh2 expression have been demonstrated in the smooth muscle cells of the murine portal veins (Ohya et al.,2002,2003). However, we only observed Kcnq1 expression, but not Kcnh2, in the developing smooth muscle component of the arterial, but not the venous, blood vessels. These data support therefore a putative role of other potassium pore-forming subunits during maturation of the venous blood vessels and/or distinct functional requirements during arterial versus venous blood vessel development.
K Channel Expression in Developing Respiratory System
In the adult trachea and airway epithelia, there are consistent evidences of potassium current involvement driving Cl− secretion as assessed by electrophysiological techniques (Grahammer et al.,2001). A putative role for Kcnq1 has been attributed (Bleich and Warh,2000; Tsevi et al.,2005); however, no data on its tissue distribution have been reported. We have observed expression of both Kcnq1 and Kcnh2 confined to the epithelium of the main respiratory airways from a very early stage of development (E10.5). These expression profiles reveal a putative role in potassium homeostasis in the developing bronchi and bronchiole almost from its early foundations. A putative role in cell proliferation can also be envisioned (Pardo,2004), although further experiments are required to clarify their role during the development of the respiratory system.
K Channel Pore-Forming Subunits in Developing Nervous System
It has been observed that mouse Kcnh2 transcripts are expressed in many neuronal tissues, of both central and peripheral developing nervous system in the adult (Polvani et al.,2003). Furthermore, it has been suggested that Kcnh2-like channels characterize the immature stage of neuronal differentiation (Arcangeli et al.,1997). However, a detailed study of the protein tissue distribution of Kcnq1 and Kcnh2 has not been described.
We have observed the expression of Kcnq1 and Kcnh2 protein in different embryonic neural tissues such as the spinal cord as well as in distinct sensory organs such as the vibrissae. However, within the developing eye and olfactory bulb, there is only expression of Kcnq1, whereas in the inner ear (cochlea), Kcnh2 is the only α-subunits expressed. These data illustrate that distinct regulatory roles for Kcnq1 and Kcnh2 seem to be played within different cell tissue layer in the developing nervous system.
The expression of potassium channel cochlear endothelium ensures the correct ion composition in the endolymph (Bleich and Warth,2000; Nicolas et al.,2001). In the adult inner ear (cochlea), Kcnq1 is the main potassium subunit responsible for endolymphatic potassium secretion (Nicolas et al.,2001) and null mutant for these gene display hearing impairment (Rivas and Francis,2005) similar to that observed in the Jervell-Lange-Nielsen syndrome. We only observed mild Kcnh2 expression in cochlea at late fetal stages (E17.5), whereas no expression of Kcnq1 is detectable. Thus, it seems that Kcnq1 expression should be established in the cochlea during postnatal stages. The presence of Kcnh2 expression in the developing and adult cochlea has not been previously reported. We can foresee nonetheless that Kcnh2 might play a role in potassium homeostasis in the endolymph, in view of its early protein expression profile in the cochlear epithelium.
Role of β-Subunits in K Channel Distribution and Function
Voltage-dependent potassium channel pore-forming subunits are modulated by distinct ancillary or β-subunits. In particular, a prominent role of the single transmembrane Kcne family β-subunits has been demonstrated for Kcnq1 and Kcnh2. At present, five different Kcne subunits have been identified (Kcne1-5), each of them playing a distinct role in Kcnq1 and Kcnh2 potassium current-mediated modulation.
In vitro experimental data using heterologous system have provided evidences that coexpression of Kcne1 with Kcnq1 generates a potassium current closely resembling the slowly activating delayed rectifier (IKs) of the cardiac action potential. Similarly, Kcne2 associated with Kcnh2 generates a potassium current closely resembling the rapidly activating delayed rectifier (IKr) current of the cardiac action potential. These observations lead to the paradigm that association of Kcnq1 + Kcne1 and Kcnh2 + Kcne2 governs the cardiac IKs and IKr repolarization currents, respectively (Nerbonne et al.,2001). However, Kcnq1 and Kcnh2 are much wider expressed than exclusively in the heart (Demolombe et al.,2001), making such a paradigm exclusively applicable to the heart. Second and more important, the complex Kcnq1/Kcne3 provides a constitutive open channel conformation when coexpressed in Xenopus oocytes and Kcne3 expression in the heart as well as in other organs is broadly documented (Franco et al.,2001b; Lan et al.,2005).
Lundquist et al. (2005) have recently provided some evidences that a fine balance of Kcne members can modulate the functional properties of Kcnq1 and Kcnh2. We have previously reported that expression of the Kcne family members is rather heterogeneous, at least during cardiogenesis, providing a suitable framework to understand the putative interactions between potassium channel pore-forming and ancillary subunits and their functional implications (Franco et al.,2001b). In this study, we have extensively analyzed the protein expression profile of Kcne3 with respect to its putative α-interacting subunits Kcnq1 and Kcnh2. Kcne3 is the only subunit that fully blocks the functional properties of Kcnq1 and Kcnh2, at least in a heterologous in vitro experimental setting (Mazhari et al.,2002). Our data demonstrate a wide variety of tissues that coexpress Kcnq1/Kcne3 or Kcnh2/Kcne3 at protein level within the same tissue layer.
These coexpression data support the notion that electrophysiological in vitro experiments using heterologous system of Kcnq1/Kcne3 and Knch2/Kcne3 do not behave similarly as in vivo conditions, and that under physiological circumstances, Kcne3 interaction with Kcnq1 and/or Kcnh2 should be tightly regulated. We can foresee three putative scenarios, either a fine protein balance between distinct Kcne family members, else a fine-tuned subcellular regionalization of the α- and β-subunits, or a posttranscriptional regulatory mechanisms that switch on/off the Kcne family member modulation of the Kcnq1/Kcnh2 pore-forming subunits. These putative control mechanisms might then be responsible for the compartimentalization of the distinct modulation of the potassium currents within distinct cell types. These hypotheses are not mutually exclusive.
The authors thank Francisco Navarro and Ricardo Caballero for critical reading of the manuscript.
- 2001. Potassium channel subunits encoded by the KCNE gene family: physiology and pathophysiology of the MinK-related peptides (MiRPs). Mol Interv 1: 95–107. , .
- 2002. Disease-associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism. FASEB J 16: 390–400. , .
- 1997. HERG- and IRK-like inward rectifier currents are sequentially expressed during neuronal development of neural crest cells and their derivatives. Eur J Neurosci 9: 2596–2604. , , , , , , , .
- 2000. The very small-conductance K+ channel KvLQT1 and epithelial function. Pflugers Arch 440: 202–206. , .
- 2004. Cardiac channelopathies: from men to mice. Ann Med 36(Suppl 1): 28–34. , , .
- 1997. Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias. EMBO J 16: 5472–5479. , , , , , .
- 1996. Developmental changes in ionic channel activity in the embryonic murine heart. Circ Res 78: 15–25. , , , , , .
- 2001. Colocalization of Kcnq1/KCNE channel subunits in the mouse gastrointestinal tract. Pflugers Arch 442: 896–902. , .
- 2001. Differential expression of KvLQT1 and its regulator IsK in mouse epithelia. Am J Physiol Cell Physiol 280: C359–C372. , , , , , , , , .
- 2001a. Divergent expression of delayed rectifier K(+) channel subunits during mouse heart development. Cardiovasc Res 52: 65–75. , , , , , , , , .
- 2001b. Methods on in situ hybridization, immunohistochemistry and beta-galactosidase reporter gene detection. Eur J Morphol 39: 3–25. , , , , .
- 2001. The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea. J Biol Chem 276: 42268–42275. , , , , .
- 2002. Kcne4 is an inhibitory subunit to the Kcnq1 channel. J Physiol 542: 119–130. , , , , , , .
- 2003. Kcne4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels. Biophys J 85: 1525–1537. , , , , , , .
- 2002. Ion channels and diseases. Med Electron Microsc 35: 117–126. , , .
- 2004. Cardiac Ikr channels minimaly comprised hERg1a and 1b subunits. J Biol Chem 22: 44690–44694. , , , , .
- 2005. Electrophysiological and molecular identification of hepatocellular volume-activated K+ channels. Biochim Biophys Acta 1668: 223–233. , , , , .
- 2005. The K+ channel KvLQT (Kcnq1) located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl− secretion. Am J Physiol Cell Physiol. 289: C564–C575. , , , , , .
- 1997. Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ Res 81: 870–878. , , , , , , , , .
- 2005. Expression of multiple KCNE genes in human heart may enable variable modulation of I(Ks). J Mol Cell Cardiol 38: 277–287. , , , , , , , , .
- 2002. Ectopic expression of Kcne3 accelerates cardiac repolarization and abbreviates the QT interval. J Clin Invest 109: 1083–1090. , , , , .
- 2001. Structural determinants of KvLQT1 control by the KCNE family of proteins. J Biol Chem 276: 6439–6444. , , , .
- 2000. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525(Pt 2): 285–298. .
- 2001. Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here? Circ Res 89: 944–956. , , , .
- 2001. Kcnq1/Kcne1 potassium channels in mammalian vestibular dark cells. Hear Res 153: 132–145. , , , , .
- 2002. Functional and molecular dentification of ERG channels in murine portal vein myocytes. Am J Physiol Cell Physiol 283: C866–C877. , , .
- 2003. Molecular variants of KCNQ channels expressed in murine portal vein myocytes: a role in delayed rectifier current. Circ Res 92: 1016–1023. , , , .
- 2004. Voltaged-gated potassium channels in cell porliferation. Physiology 19: 285–292. .
- 2003. Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3. Gene Expr Patterns 3: 767–776. , , , , , , , , .
- 1999. Voltage-gated potassium channels: from hyperexcitability to excitement. FEBS Lett 452: 31–35. .
- 1999. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci 868: 344–355. , , , , , , , , .
- 2005. Inner ear abnormalities in a Kcnq1 (Kvlqt1) knockout mouse: amodel of Jervell and Lange-Nielsen síndrome. Otol Neurotol 26: 415–424. , .
- 2005. Voltage-gated ion channels. Curr Biol 15: R44–R47. , , .
- 1999. Structure and function of cardiac potassium channels. Cardiovasc Res 42: 377–390. .
- 2004. Pharmacology of cardiac potassium channels. Cardiovasc Res 62: 9–33. , , , , .
- 2003. Novel gene hKcne4 slows the activation of the Kcnq1 channel. Biochem Biophys Res Commun 303: 808–813. , , , , , , , .
- 2005. Kcnq1/Kcne1 channels during germ-cell differentiation in the rat: expression associated with testis pathologies. J Cell Physiol 202: 400–410. , , , , , , , .
- 2004. Homing of intestinal immune cells. Novartis Found Symp 263: 179–188. , , .
- 2004. Renal potassium channels: recent developments. Curr Opin Nephrol Hypertens 13: 549–555. .
- 2002. The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse. Pflugers Arch 443: 822–828. , , , , , , , , .