INTRODUCTION

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

POTASSIUM CHANNELS are found in virtually all mammalian cells. They form the most diverse group of ion channels (~80 pore-forming subunit genes) that can be divided into three main structural classes comprising two, four, or six transmembrane segments. All these K⁺ channel subunits have in common a conserved motif called the P domain, which is part of the K⁺-selective filter that provides the specificity to K⁺ transport. In addition to the pore-forming subunits themselves, K⁺ channels comprise in their structure associated modulatory subunits designated as -subunits. They are usually not essential for the formation of the ionic pore, but they determine the stability of the channel complex in the membrane and    modulate biophysical, regulatory, and pharmacological properties (18).

KCNE1, also named IsK or minK, belongs to a family of small transmembrane proteins (KCNE1, -2, -3, and -4 and KCNE1L). Originally KCNE1 was cloned from a rat kidney library and expressed in Xenopus laevis oocytes, leading to a slowly activating K⁺ current (I_Ks) (70). However, KCNE1 has been regarded as somewhat of an enigma in the ion channel field because it has none of the hallmarks of conventional K⁺ channels, particularly the P domain. Moreover, it was found that the amplitude of KCNE1 currents in oocytes saturates at low levels of cRNA injections (9), and attempts to express KCNE1 currents in numerous eukaryotic cells failed (42). These observations indicated the lack of an essential cofactor in these cells. The explanation of this phenomenon is the coassembly of KCNE1 with a Shaker-type K⁺ channel -subunit, KCNQ1 (also named KvLQT1), identified by positional cloning in patients with long QT syndrome (6, 17, 61). KCNQ1 exists as an endogenous X. laevis KCNQ1 in oocytes (61). The assembly with KCNE1 increases the voltage dependence and current amplitude of KCNQ1, slows down activation kinetics, and changes pharmacological properties (Fig. 1, A and B) (10). Mutations in both genes are associated with a hereditary form of cardiac arrhythmia, so-called long QT syndromes (7, 38, 55). Monoallelic mutations in either gene cause the dominant form of the syndrome, called Romano-Ward syndrome (RWS; long QT syndrome type 5), a life-threatening disease characterized by prolonged cardiac repolarization and polymorph ventricular arrhythmias. Biallelic mutations lead to the Jervell and Lange-Nielsen syndrome (JLNS), with a severe long QT phenotype associated with profound bilateral deafness (51, 73). In the case of the RWS, mutations in other ion channel genes have also been described (for review, see Refs. 38, 57). These genes include the Na⁺ channel gene SCN5A and two K⁺ channel genes, KCNH2 (HERG) and KCNE2 (MiRP1), the latter encoding a protein similar to KCNE1. In addition, two other gene loci have been described that correspond to a ryanodine receptor (RYR2) on chromosome 1q42 (56) and a yet unknown gene on 4q25 (62).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   KCNE1 coassembles with KCNQ1. A: putative membrane topology of KCNQ1 and KCNE1. KCNQ1 consists of 6 transmembrane domains and 1 P loop between S5 and S6. KCNE1 has only 1 transmembrane domain with the NH2 terminus facing the extracellular side. B: effect of KCNE1 on KCNQ1 current. COS cells transfected with KCNQ1 alone (Q1, top trace) or cotransfected with KCNQ1 and KCNE1 (Q1 + E1, bottom trace) were examined in whole cell mode (clamp protocol: 80, +50, 40 mV). Q1 transfection led to voltage-activated K+ outward current. Cotransfection with Q1 and E1 enhanced current amplitude and voltage dependence and slowed down activation kinetics. B is a kind gift from Georges Romey, Sophia Antipolis, France (published with permission).

Besides its assembly with KCNQ1, KCNE1 was also shown to associate with KCNH2 (48) and Cl channels (4). However, these two types of interactions are less documented than the one with KCNQ1 and still await confirmation. On the other hand, it is clear that in addition to KCNE1, both KCNE2 (71) and KCNE3 (63) can interact with KCNQ1 to form K⁺ channels with specific biophysical properties (for review, see Ref. 60).

The KCNE1/KCNQ1 channel complex is abundant in heart muscle, inner ear, and a variety of epithelial tissues (Fig. 2) (8, 65). The generation of a null mutant mouse for the KCNE1 allows the detailed in vivo exploration of the physiological roles of this specific channel in cardiac as well as noncardiac tissues. In humans the KCNE1 gene is located on chromosome 21q22.1-q22.2 (15) and in mice on chromosome 16 (64.4 cM) (Ref. 37). For construction of the knockout mouse the complete coding sequence (located on exon 2) was deleted and replaced by the neomycin resistance gene (75). In another KCNE1 knockout mouse model, in addition to deletion of the KCNE1 coding sequence, lacZ and neomycin resistance genes were inserted (40). Moreover, a spontaneous mutation leading to a truncated protein (66 instead of 129 amino acids) has been reported (43).

View larger version (73K): [in this window] [in a new window]   Fig. 2.   Distribution of KCNQ1 and its -subunits KCNE1, -2, and -3 in mouse tissues. Besides in the heart, KCNQ1 is predominantly expressed in epithelial tissues. KCNE1 was highly abundant in heart and kidney. The strong expression in mouse stomach is in contrast to human tissue, in which we could not detect KCNE1 (31). KCNE2 is strongly expressed in stomach and eye, and KCNE3 is strongly expressed in the intestinal tract. In contrast to recent studies (1, 2), we failed to detect KCNE2 and KCNE3 (63) in heart and skeletal (sk) muscle, respectively. The primers were as follows: KCNQ1 sense 5'-CTGAGAAAGATGCGGTGAAC-3', antisense 5'-TGGGGGTCAGCAGTGTCTCC-3'; KCNE1 sense 5'-CGACTGTTCTGCCCTTTCTG-3', antisense 5'-CTCAGTGGTGCCCCTACAAT-3'; KCNE2 sense 5'-GAGGAGGAACACAACAGC-3', antisense 5'-CCAGGTTCTCATGGATGG-3'; KCNE3 sense 5'-AGCTCTTCCCATACCTCAAT-3', antisense 5'-AATCCTCTTACCAGTTTCCT-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 5'-GTGCTGGGCTACCTGCTCTA-3', antisense 5' TCGTCCTTGTCTTTCTTCAC 3'. The PCR was made under standard conditions using 32 cycles. Specific KCNQ1 and KCNE1, -2, and -3 PCR products were recognized by Southern hybridization with internal oligonucleotides. E16-18, embryonic days 16-18.

The clinical relevance of KCNE1 mutations in humans is known for heart and inner ear. Here we give an overview on the multitissue phenotype of the KCNE1 knockout mouse, which could have an impact for human pathophysiology and disease.

	KCNE1 KNOCKOUT MICE DISPLAY A MILD CARDIAC PHENOTYPE

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

Physiologically, repolarization of heart action potential is dependent on several K⁺ conductances, including KCNE1/KCNQ1. The cardiac KCNE1/KCNQ1 current (I_Ks) is activated via depolarization during the action potential and shows slow activation kinetics. In mouse heart, KCNE1 is strongly expressed with some decay during the first weeks after birth (25, 27). With PCR methods, we find KCNE1 abundant in both atrium and ventricle (Fig. 2). -Galactosidase activity, which was under control of the KCNE1 promoter, indicates a strong and specific expression of KCNE1 in cells of the sinus node and atrioventricular node, in lower right atrial septum, and in the proximal conducting system. In the ventricle, cells belonging to the conducting system are also stained (40).

Does KCNE1 gene disruption affect the heart action potential? In one study microelectrode measurements show no difference in action potential duration between knockout and wild-type mice (13). In electrocardiogram recordings, there is no direct correlate of the QT prolongation observed in JLNS patients: QT interval is very similar in both genotypes under control condition and in the presence of isoproterenol stimulation (25, 40). However, the QT-RR adaptation is significantly exacerbated in KCNE1 knockout mice, leading to a prolonged QT interval during bradycardia (25). A similar increased QT-RR adaptability is described for long QT patients (33, 50). What, then, is the explanation for the shorter QT intervals in knockout mice at high heart rates? One might speculate that KCNQ1 alone, which is still present in KCNE1 knockout mice, could lead to a fast-activating repolarizing K⁺ current at high heart rates. Furthermore, secondary compensatory effects, i.e., via differences in plasma K⁺ or high aldosterone, are possible explanations.

Taken together, the KCNE1 knockout mouse is an interesting model for JLNS, but with clear limitations due to species differences. Concerning the localization of KCNE1 in pacemaker cells and the conducting system, especially in the lower right atrium, it is speculated that KCNE1 might play a role in common reentrant arrhythmias such as atrioventricular nodal reentrant tachycardia and common atrial flutter (40). Further studies are needed to elucidate the underlying mechanisms and a possible role of KCNE1 in more detail.

	KCNE1 GENE KNOCKOUT IS ASSOCIATED WITH DEAFNESS AND SHAKER-WALTZER BEHAVIOR

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

Endolymph is an extracellular fluid with a unique composition, a high K⁺ concentration (150 mmol/l) and a low Na⁺ concentration (4 mmol/l). The ionic composition is crucial for signal transduction of sensory hair cells of the cochlear duct and the vestibular labyrinth. Numerous genes are known to cause deafness (http://www.uia.ac.be/dnalab/hhh/). Among these, some genes for membrane transporters and ion channels have been identified that are involved in the complex mechanisms (69, 76) of endolymph secretion and generation of the endocochlear potential: Na⁺-2Cl-K⁺ cotransporter (21, 24, 28), H⁺ ATPase (36), and KCNE1/KCNQ1 K⁺ channels. KCNE1 and KCNQ1 are localized in the luminal membrane of endolymph-producing marginal cells from the stria vascularis and of vestibular dark cells (11, 41, 52, 52, 59, 75) representing the efflux pathway for K⁺. Their regulation by purinergic (47), adrenergic (78), and muscarinic (77) receptors and cAMP (67, 68) and their pharmacology (64) have been described in detail.

Already in the 19th century cases of sudden death combined with deafness were reported (49) probably corresponding to JLNS. Like patients suffering from JLNS, KCNE1 knockout mice are also profoundly and bilaterally deaf and exhibit an obvious vestibular dysfunction, leading to rapid head bobbing and bidirectional circling, which is referred to as Shaker-Waltzer behavior (23, 40, 43, 75).

In the cochlea of the inner ear the position of Reissner's membrane is dependent on the balance between endolymph production and reabsorption. In KCNE1 knockout mice, Reissner's membrane collapses postnatally, indicating that K⁺ secretion and concomitant water flux are strongly reduced. This impaired endolymph production leads to cell death of sensory hair cells and, within 6 wk, to degeneration of the majority of the spiral ganglion neurons (75). Interestingly, the cell layers of stria vascularis show slight morphological changes, namely an enlargement of the intercellular space in the intermediate cell layer, which corresponds to fluid waiting to be secreted. The endolymph-producing marginal cells appear to be grossly normal (75). In the vestibular labyrinth the vestibular wall collapses, similar to Reissner's membrane in the cochlea. Within 6-7 mo after birth, the sensory hair cells of the vestibular system degenerate and disappear. Endolymph-producing vestibular dark cells of KCNE1 knockout mice are larger but do not undergo cell death (52). In Ussing chamber experiments short-circuit current as a measure of secretion is almost completely abolished in KCNE1 knockout mice (75). Interestingly, KCNQ1 immunostaining of the luminal membrane of dark cells disappears in KCNE1 / mice, indicating that KCNE1 is essential for KCNQ1 membrane targeting and/or stability of KCNQ1 in the membrane (Fig. 3) (52). It is not presently known if KCNE1 also plays such a trafficking role in other organs where it is associated with KCNQ1. More recently, it was shown that KCNQ1 knockout mice present similar inner ear pathology (11, 41).

View larger version (115K): [in this window] [in a new window]   Fig. 3.   Immunolocalization of KCNE1 and KCNQ1 in the vestibular system. A: KCNE1 staining of a KCNE1 wild-type vestibular crista. The endolymphatic space is located at top right. At the base of each side of the crista, the luminal membrane of dark cells is strongly stained (arrows). Other parts of the crista, sensory and transitional epithelium, are not labeled. B: KCNQ1 staining of a KCNE1 wild-type vestibular crista. As with KCNE1 protein, a strong luminal staining of KCNQ1 (arrows) was detected. C: KCNQ1 staining of a KCNE1 knockout vestibular crista. Compared with wild type in B, the KCNQ1-specific staining is lost. Please note the changes in morphology and the collapse of the endolymphatic space (*). Figure 3 is a kind gift from Marie-Thérèse Nicolas and Danielle Demêmes, Monpeiller, France (published with permission).

In conclusion, the heteromultimeric KCNE1/KCNQ1 channel plays a key role for physiological endolymph secretion, which is a prerequisite for signal transduction in cochlea and vestibular system. In addition, KCNE1 is essential for normal KCNQ1 localization and function in the inner ear.

	ROLE OF KCNE1 FOR RENAL SALT AND WATER REABSORPTION

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

In mammalian kidney, KCNE1 is predominantly expressed in proximal tubules. Immunofluorescence experiments reveal a colocalization of KCNE1 and KCNQ1 proteins in the brush-border membrane (22, 66, 74). However, a weaker expression of KCNE1 and KCNQ1 in other nephron segments is not excluded by these experiments.

Is renal function affected by KCNE1 gene knockout? Interestingly, KCNE1 / mice are hypokalemic and exhibit as a sign of dehydration an increased hematocrit, suggesting an impaired renal electrolyte balance and enhanced water loss (Fig. 4) (3, 74). The inulin clearance as a measure of glomerular filtration rate is not changed; however, the fractional excretion of NaCl and fluid is markedly increased. Micropuncture experiments reveal a reduced K⁺ concentration in late proximal and early distal tubular fluid of knockout mice due to a reduced proximal tubular K⁺ efflux through luminal KCNE1 K⁺ channels (74).

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Localization and function of KCNE1 in the kidney. A-D: immunolocalization of KCNE1 and KCNQ1. A: KCNE1 staining (green) was most prominent in brush-border membrane of proximal tubules. Hoe-33342 nucleus staining is shown in blue, and differential interference contrast picture is in gray scale. B: no KCNE1-specific staining was observed in KCNE1 knockout (/) mice. KCNQ1 (red) was almost absent in S1 segments of proximal tubules (*; C) but colocalized with the KCNE1 in the brush-border membrane of S2 and S3 segments (D). Interestingly, KCNQ1 brush-border localization was not affected in KCNE1 knockout mice. Inulin clearance experiments revealed an increased fractional urinary Na+ excretion (FeNa+; E) and a tendency to lose K+ (F). The loss of salt and water led to an enhanced hematocrit (G) as a means for volume depletion. Fecal and urinary K+ loss induced a relative hypokalemia (H). Data were taken from inulin clearance experiments under control conditions (74). In the same study, the relative hypokalemia was not observed after long-lasting micropuncture experiments; however, another study using a larger number of animals without pretreatment [KCNE1 knockout 3.77 ± 0.12, KCNE1 wild-type mice 4.48 ± 0.08 mmol/l (3)] confirmed the reduced plasma K+ of KCNE1 knockout mice. FeK+, fractional urinary K+ excretion.  P < 0.05 vs. KCNE1 wild-type (+/+) mice.

Proximal tubules physiologically reabsorb Na⁺ and substrates using Na⁺/H⁺ exchange (NHE3) and Na⁺-coupled glucose and amino acid transport systems. This Na⁺-coupled transport depolarizes the luminal membrane, thereby reducing the driving force for further transport. Thus luminal K⁺ channels are required to repolarize the luminal membrane. In studies on isolated in vitro perfused proximal tubules of KCNE1 / mice, phenylalanine and glucose in the luminal fluid led to an enhanced depolarization of the membrane voltage compared with wild-type mice. The gene knockout could be mimicked by addition of the K⁺ channel inhibitor Ba²⁺ to the luminal fluid in perfused proximal tubules of wild-type mice (74). This loss of driving force for substrate reabsorption explains the increased fractional glucose excretion of KCNE1 / mice. To exclude additional effects of the knockout on distal nephron segments, amiloride can be used as a pharmacological tool to assess Na⁺ reabsorption via epithelial Na⁺ channels (ENaC). Interestingly, amiloride gives rise to a higher Na⁺ excretion in knockout mice, which argues against an impaired reabsorption of distal nephron segments.

Taken together, these data indicate an important role of the KCNE1/KCNQ1 complex as a luminal K⁺ channel in mouse proximal tubules. This K⁺ conductance located in the brush-border membrane grants the driving force for electrogenic Na⁺ and substrate reabsorption.

	ALDOSTERONE SECRETION IS REGULATED BY KCNE1-DEPENDENT K+ CONDUCTANCE

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

Under normal diet, KCNE1 / mice exhibit hemoconcentration and hypokalemia. Probably, the hypokalemia is mostly due to an increased plasma aldosterone concentration (Fig. 5). Interestingly, plasma renin concentrations under normal diet are similar in both wild-type and knockout mice, indicating that the increase in aldosterone is not due to enhanced renin concentration. In KCNE1 knockout mice high aldosterone stimulates Na⁺ reabsorption in distal colon, paralleled by enhanced K⁺ secretion and fecal K⁺ loss. Also, renal fractional K⁺ excretion is enhanced but does not reach significance. Low-Na⁺ diet increases and low-K⁺ diet reduces aldosterone plasma concentrations in both genotypes in a similar way. In contrast, under high-K⁺ diet, aldosterone is approximately fivefold higher in KCNE1 / mice, although plasma K⁺ concentration is still lower than in wild-type mice (3). The explanation for this surprising finding is the expression of KCNE1 in aldosterone-producing adrenal glomerulosa cells. Physiologically, aldosterone secretion is activated via two major stimuli: high plasma K⁺ concentration and ANG II, both finally leading to the activation of depolarization-activated T-type Ca²⁺ channels. This Ca²⁺ influx then in turn activates aldosterone secretion. The K⁺ conductance in glomerulosa cells comprises at least two types of K⁺ channels. ANG II inhibits one type, namely TASK1 (19), via Ca²⁺/calmodulin-dependent protein kinase II and shifts the voltage dependence of the T-type Ca²⁺ channel to more hyperpolarized values (14). On the other hand, even small increases in plasma K⁺ suffice to depolarize the cell, thereby activating Ca²⁺ influx. This depolarization is thought to activate voltage-dependent K⁺ channels, which then limit the Ca²⁺ influx and aldosterone secretion (45). The impressive effect of high-K⁺ diet in KCNE1 / mice, together with the increased aldosterone under normal K⁺ diet without a concomitant increase in renin, indicates a crucial role of KCNE1 for repolarization of glomerulosa cells. A portion of the increase in aldosterone under high-K⁺ diet is caused via renin. The elevated renin is probably not due to a direct effect of the KCNE1 gene deletion on renin-producing cells because they do not express KCNE1. The mechanism by which renin is increased remains to be elucidated. Taken together, these data suggest a concerted mechanism of action of the increased renin/ANG II concentration and enhanced K⁺ sensitivity of glomerulosa cells in KCNE1 / mice.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effect of KCNE1 gene disruption on aldosterone secretion. KCNE1 knockout mice had an increased plasma aldosterone concentration under normal diet (containing 0.9% K+) compared with wild-type animals. Interestingly, plasma renin concentration was similar for both genotypes under these conditions. At high-K+ diet (3%), aldosterone was 5-fold increased in knockout mice compared with wild-type mice, paralleled by a 3-fold increase in renin. Under low-K+ diet (0.05%) and low-Na+ diet (0.01%), aldosterone and renin concentrations were not different among the genotypes (data from Ref. 3).

Further studies are required to investigate the impact of these data on human pathology. Both low plasma K⁺ and high aldosterone concentrations are possibly harmful: the occurrence of life-threatening torsades de pointes arrhythmias in patients suffering from long QT syndromes is especially high during hypokalemia (26, 58). On the other hand, aldosterone was shown to have a deleterious effect on the progression of chronic heart failure (54).

	KCNE1 IN EXOCRINE PANCREAS

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

KCNE1 and KCNQ1 are abundant in pancreatic acinar cells (22, 70, 80), leading to a voltage-dependent and slowly activating K⁺ current (39). In wild-type mice, KCNQ1 seems to be mainly located in the basolateral membrane. However, it cannot be excluded that KCNQ1 is present to a weaker extent in luminal and vesicular membranes. Interestingly, in KCNE1 / mice, KCNQ1-specific immunofluorescence is less focused on the basolateral membrane and also present in the cytosol, suggesting an impaired membrane targeting of KCNQ1 (79). In addition, the pancreatic secretory granules are irregularly distributed in KCNE1 / mice: some acini are completely packed with granules, whereas other acini are almost without any secretory granules. Unfortunately, the pathophysiological mechanisms underlying this phenomenon are not understood.

The biophysical properties of the KCNE1/KCNQ1 K⁺ current in pancreatic acini resemble very much the cardiac KCNE1/KCNQ1 channel complex (6, 61) and the voltage-dependent current observed in adrenal glomerulosa cells (6, 45). This component of whole cell K⁺ current is strongly augmented in the washout phase after cholinergic stimulation when the intracellular Ca²⁺ activity and the Ca²⁺-activated Cl conductance are already decreased. In pancreatic acinar cells of KCNE1 / mice, this current is almost completely abolished, indicating, together with the histological findings, a functional role of KCNE1 in the KCNQ1 channel complex in rodent pancreas (79). Further studies are needed to elucidate the localization and physiological role of KCNE1 during electrolyte and enzyme secretion.

	INTESTINAL ION TRANSPORT IS ALTERED IN KCNE1 / MICE

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

In metabolic cage experiments KCNE1 / mice lose an impressive amount of Na⁺ and K⁺ with feces compared with wild-type mice (3). The loss of K⁺ might be explained by an increased secretion in distal colon due to stimulated aldosterone secretion in knockout mice. In fact, we observe in KCNE1 / mice threefold increased amiloride-sensitive Na⁺ reabsorption in Ussing chamber experiments of distal colon. Intriguingly, this observation cannot explain the increased Na⁺ loss via feces in metabolic cages, but one would expect the opposite, namely a reduced loss of Na⁺. One might speculate that similar to Na⁺ reabsorption in renal proximal tubules, KCNE1 plays a role in Na⁺ and substrate reabsorption in proximal parts of small intestine. However, we are not able to detect a specific immunofluorescence, possibly due to an amount of KCNE1 protein below our detection limit (79). In contrast to immunofluorescence, Northern blot analysis reveals an expression of KCNE1 in rat duodenum (70). Such a role of KCNE1 in duodenal Na⁺-coupled transport together with the aldosterone-stimulated K⁺ secretion in distal colon could explain the combined fecal loss of Na⁺ and K⁺ in knockout mice. Theoretically, an enhanced Na⁺ content of pancreatic/intestinal secretion could also cause the increased fecal Na⁺ loss. Thus far, however, there is no experimental evidence supporting this hypothesis.

	KCNE1 IN AIRWAY EPITHELIUM

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

There is controversy in discussions of expression and function of KCNE1 in airway epithelium. In two studies a basolateral K⁺ conductance (35) activated during regulatory volume decrease (44) is reported to be KCNE1 dependent. In contrast, we find no expression of KCNE1, but do find KCNE3, in murine trachea. In KCNE1 knockout mice, electrogenic cAMP-mediated Cl secretion, which requires basolateral K⁺ channels to provide the driving force, is higher in KCNE1 knockout mice (22, 32). Na⁺ reabsorption via epithelial Na⁺ channels is slightly higher in KCNE1 knockout mice. One possible explanation for these differences might be the altered hormone status after KCNE1 gene disruption, i.e., the enhanced aldosterone concentration (3). We conclude from these data that, at least in mouse trachea, Cl secretion and Na⁺ reabsorption do not require KCNE1.

	GASTRIC ACID SECRETION REQUIRES KCNQ1 BUT NOT KCNE1

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

KCNQ1 mRNA is abundant in gastric mucosa (16, 80), suggesting a possible role of KCNQ1 for gastric ion transport. Interestingly, KCNQ1 colocalizes with the gastric H⁺-K⁺-ATPase in the tubulovesicular system of the luminal membrane compartment (20, 31). Inhibition of KCNQ1 by the chromanol 293B almost completely abolishes acid secretion (31), and the KCNQ1 gene disruption leads to a loss of acid secretion and gastric hyperplasia in knockout mice (41). These results indicate a crucial role of KCNQ1 for luminal K⁺ recycling during acid secretion. In rodent stomach KCNE1, -2, and -3 (all putative KCNQ1 -subunits known so far) are expressed, with the highest levels of expression for KCNE2 (20, 22, 31, 70). Localization of KCNE2 in parietal cells by in situ hybridization (20) and activation of KCNQ1/KCNE2 channels by acidic extracellular pH (31), cAMP, and inositol 1,4,5-trisphosphate/Ca²⁺ (unpublished data) make KCNE2 the likely -subunit of KCNQ1 in parietal cells. We exclude a major role of KCNE1 for H⁺ secretion in rodents because the KCNE1 gene disruption neither reduces acid secretion nor changes the effect of the KCNQ1 inhibitor 293B (31). In human stomach KCNE1 is not expressed, supporting the hypothesis that KCNE2 and/or KCNE3 coassemble with KCNQ1 to form the luminal K⁺ conductance of parietal cells.

	KCNE1 KNOCKOUT MICE ACCUMULATE MATURE T LYMPHOCYTES

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

In mouse thymus both KCNE1 and KCNQ1 are weakly expressed. They are not detected in this tissue by PCR techniques using 32 cycles (Fig. 2) but are detected with 40 cycles (12). Interestingly, KCNE1 gene disruption leads to accumulation of mature T cells in thymus and peripheral lymphoid tissue of adult mice. However, the molecular mechanisms underlying this accumulation and the possible functional modulation of the immune system by KCNE1 need to be elucidated (12).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION KCNE1 KNOCKOUT MICE DISPLAY... KCNE1 GENE KNOCKOUT IS... ROLE OF KCNE1 FOR... ALDOSTERONE SECRETION IS... KCNE1 IN EXOCRINE PANCREAS INTESTINAL ION TRANSPORT IS... KCNE1 IN AIRWAY EPITHELIUM GASTRIC ACID SECRETION REQUIRES... KCNE1 KNOCKOUT MICE ACCUMULATE... CONCLUSIONS REFERENCES

The KCNE1 knockout mouse displays a very complex phenotype arising from direct effects due to the loss of the KCNE1 protein and due to indirect compensatory mechanisms (Table 1). In most tissues KCNE1 probably coassembles with KCNQ1; however, one has to be aware of other partner proteins. In heart muscle the loss of KCNE1/KCNQ1 (6, 61) and KCNE1/KCNH2 (4, 48) interaction, leaving homomeric KCNQ1 and KCNH2 (HERG) channels behind (or these channels associated with alternative partners), might explain the pronounced QT-RR adaptability.

Like patients suffering from homozygous KCNE1 mutations (JLNS), the mice are profoundly deaf. In addition, knockout mice suffer from severe disturbance of the vestibular system, showing head bobbing and bidirectional circling (Shaker-Waltzer behavior). In JLNS patients other K⁺ channels and/or secondary mechanisms probably largely compensate for vestibular defects.

Concerning the cardiac phenotype, the KCNE1 knockout mouse is an interesting animal model for JLNS, offering the possibility of extensive physiological and pharmacological experiments. Despite the fact that there are evident limitations of this model, which are mostly due to species differences in terms of heart rate, heart size, and different levels of expression of ion channels, the KCNE1 knockout mouse can help to obtain new insights in pathophysiology and disease-related phenomena.

The data on renal ion and glucose transport suggest a functional coupling of Na⁺ and glucose reabsorption to a luminal KCNE1-dependent K⁺ conductance. Because many transport mechanisms in small intestine are similar to those in renal proximal tubules, the KCNE1 knockout mouse can be a useful tool to investigate the possible role of KCNE1 for duodenal glucose and amino acid reabsorption.

The new observations on the role of KCNE1 for aldosterone secretion in glomerulosa cells and plasma K⁺ homeostasis might be of great importance for the treatment of JLNS patients because life-threatening torsades de pointes arrhythmia often occurs during hypokalemia. These patients could benefit from a slight increase in plasma K⁺, i.e., via administration of spironolactone.

The present studies on the KCNE1 knockout mouse have provided new knowledge on the pathophysiology of a gene whose human disease correlate was supposed to be well understood. These findings and future studies will help to gain a more comprehensive understanding of KCNE1 physiology and perhaps a gene-specific treatment of patients.