Tetrodotoxin-blockable depolarization-activated Na+ currents in a cultured endothelial cell line derived from rat interlobar arter and human umbilical vein

Voltage-dependent Na⁺ current (I_Na) was identified in cultured endothelial cells derived from rat interlobar artery (RIAE cells) and human umbilical vein (HUVE cells). Tetrodotoxin (TTX) reduced I_Na in a dose-dependent manner with the apparent dissociation constant (K_d) of 1.4 M. Low sensitivity of I_Na to TTX as well as its kinetics and voltage-dependent properties indicates that voltage-gated Na channels expressed in vascular endothelial cells belong to the so-called TTX-resistant type [2].     

Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve

Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na⁺. Relief of TTX blockage was also observed by adding 100 mM chloride salts of Li⁺, hydrazine⁺, guanidine⁺ and K⁺ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.     

Assessment of candidate ocular biomarkers of ageing in a South African adult population: Relationship with chronological age and systemic biomarkers

Certain anatomic and functional parameters of the eye change with increasing chronological age. They may, therefore, serve as potential biomarkers of ageing. We investigated associations between four such ocular parameters (lens density, retinal vessel calibre, corneal endothelial cells and retinal nerve fibre layer thickness) and two ‘cellular’ biomarkers of ageing (leukocyte telomere length and CDKN2A expression), with frailty (a clinical correlate of biological ageing) in a population of South African adults. All ocular parameters revealed an association with either telomere length or CDKN2A expression. However, lens density was most strongly correlated with age, increased CDKN2A expression, and with frailty (p = 0.05 and 0.03, respectively). Narrow retinal arteriolar diameter, associated with increased chronological age, was also associated with increased CDK2NA expression (0.42 vs. 0.31, p = 0.02) but not with frailty. Ocular parameters may aid in determining biological age, warranting investigation in longitudinal studies.     

Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Nav1.9, in mouse dorsal root ganglion neurons

Small dorsal root ganglion neurons express preferentially the Na⁺ channel isoform Na_V1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na⁺ current. We investigated properties of the Na⁺ current mediated by Na_V1.9 (I_NaN) using the whole-cell, patch-clamp recording technique. To isolate I_NaN from heterogeneous TTX-R Na⁺ currents that also contain another type of TTX-R Na⁺ current mediated by Na_V1.8, we used Na_V1.8-null mutant mice. When F^– was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for I_NaN shifted in the hyperpolarizing direction with time. Such a time-dependent shift of the kinetics was not observed when Cl^– was used as an internal anion. Functional expression of I_NaN declined with time after cell dissociation and recovered during culture, implying that Na_V1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of I_NaN increased dramatically after a variable delay, as if inactive or silent channels had been kindled. Such an unusual increase of the amplitude could be prevented by adding ATP to the pipette solution or by recording with the nystatin-perforated patch-clamp technique, suggesting that the rupture of patch membrane affected the behaviour of Na_V1.9. These peculiar properties of I_NaN may provide an insight into the plasticity of Na⁺ channels that are related to pathological functions of Na⁺ channels accompanying abnormal pain states.     

Involvement of p27(KIP1) in proliferation of the retinal pigment epithelium and ciliary body

According to observations in various cell lines, elimination of the cyclin-dependent kinase inhibitor p27(KIP1) during the late G1 phase of the cell cycle is required for progression to the S phase. Eyes from C57BL/6 mice at embryonic days 13, 14, and 18, and at 4 weeks of age, were analyzed by a bromodeoxyuridine cell proliferation assay and by immunocytochemistry using anti-p27(KIP1) antibody. On embryonic days 14 and 18, p27(KIP1) was detected in the ciliary body. This protein also was detected in the nuclei of the many cells of the retinal pigment epithelium on embryonic day 18, and was present in all such cells at 4 weeks of age. When p27(KIP1)-/- knockout and control mice were injected with bromodeoxyuridine between postnatal days 7 and 10 and analyzed on day 11, positive cells were abundant in the retinal pigment epithelium and the ciliary body of p27(KIP1)-/- mice, whereas few cells were positive in control mice. By fluorescent nuclear staining in whole mounts of retinal pigment epithelium at 12 weeks of age, more nuclei were present in p27(KIP1)-/- than in the wild-type mice. These results suggest that p27(KIP1) was involved in regulation of proliferation in the RPE and the ciliary body.     

K+-Stimulated Na+ transport in frog-skin epithelia

Increasing [K⁺] from 2.5 mmol/l to 115 mmol/l on the serosal side of the frog skin produces a rapid decrease of short-circuit current (I_sc) that is followed, within a few minutes, by a recovery of I_sc to near or above its control value. After isolation of the epithelium by a procedure involving collagenase treatment and physical removal of thecorium, increasing serosal [K⁺] still produced a depression of I_sc but no significant recovery phase. By itself, collagenase treatment reduced but did not eliminate the recovery phase. The recovery phase was also markedly depressed by the beta-adrenergic blocker oxprenolol, but not by propranolol, atropine or indomethacin. Amiloride, given during the recovery phase, caused I_sc to reverse to a small outward value. These results suggest that the recovery phase of I_sc seen in the response to increased serosal [K⁺] represents an increase in Na⁺ influx through amiloride-sensitive channels which is triggered by the release of an intermediary agent, possibly a beta-adrenergic agonist, from some structure in thecorium.     

Modification of cloned brain Na+ channels by batrachotoxin

The effects of batrachotoxin (BTX) on cloned α-subunit Na⁺ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na⁺ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×10⁶ mol⁻¹·s⁻¹ at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The K _D value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (K _D=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH₃ cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na⁺ channels.     

成年大鼠眼睫状体缘色素上皮组织的神经前体细胞的培养和鉴定

目的　探讨成年大鼠眼睫状体缘色素上皮产生神经前体细胞的潜力及其生物学特征。方法　取成年SD大鼠睫状体缘处的色素上皮组织块 ,置于含bFGF和B2 7的DMEM/F12 无血清培养液中进行神经前体细胞培养 ,免疫组化反应染色鉴定细胞的表型。结果　培养 5～ 8d ,组织块长出由众多无色素和色素细胞构成的细胞集落 ,集落的大多数细胞处于增殖状态(BrdU反应阳性 ) ,并表达神经前体细胞的标志物 (nestin)。撤掉bFGF和B2 7,加入胎牛血清培养 3～ 5d ,集落的细胞发生分化 ,分别表达神经元和星形胶质细胞的标志物 (NSE、GFAP) ,但不表达少突胶质细胞的标志物 (O4 )。结论　由成年大鼠睫状体缘色素上皮长出的细胞集落不仅含有增殖能力和多分化潜能的神经前体细胞 ,而且尚含有色素细胞 ,该部位可能是视网膜神经感觉层和色素上皮共同保守的细胞生发带     

Carbodiimide modification reduces the conductance and increases the tetrodotoxin sensitivity in batrachotoxin-modified sodium channels

The relationship between the channel entrance and the tetrodotoxin (TTX) binding site was investigated by chemical modification at the extracellular surface of bilayer-incorporated batrachotoxin-(BTX) modified sodium channels using an impermeant carbodiimide in the presence or absence of exogenous nucleophiles. Two (classes of) groups could be modified such that the open-channel conductance was decreased while TTX binding was unaffected, and TTX did not protect against this modification. Because the final conductance level depends on the exogenous nucleophile, each covalent modification appears to involve a carboxyl group. In addition, a third (carboxyl) group could be modified such that TTX binding affinity was increased. These results suggest that the channel entrance and the TTX binding site are spatially separate, which supports previous suggestions that the mechanism by which guanidinium toxins close sodium channels involves a conformational change subsequent to toxin binding.     

Pharmacological properties of neuronal TTX-resistant sodium channels and the role of a critical serine pore residue

Voltage-gated sodium channels can be characterized by their sensitivity to inhibitors. Na_v1.5 is sensitive to block by cadmium and extracellular QX-314, but relatively insensitive to tetrodotoxin and saxitoxin. Na_v1.4 is tetrodotoxin- and saxitoxin-sensitive but resistant to cadmium and extracellular QX-314. Na_v1.8 and Na_v1.9 generate slowly inactivating (I_TTXr-Slow) and persistent (I_TTXr-Per) currents in sensory neurons that are tetrodotoxin-resistant. Tetrodotoxin sensitivity is largely determined by the identity of a single residue; tyrosine 401 in Na_v1.4, cysteine 374 in Na_v1.5 and serine 356 and 355 in Na_v1.8 and Na_v1.9. We asked whether Na_v1.8 and Na_v1.9 share other pharmacological properties as a result of this serine residue. I_TTXr-Slow and I_TTXr-Per were saxitoxin-resistant and resistant to internal QX-314. I_TTXr-Slow was also resistant to external QX-314 and displayed a approximately fourfold higher sensitivity than I_TTXr-Per to cadmium. The impact of the serine residue was investigated by replacing tyrosine 401 in Na_v1.4 with serine (Y401S) or cysteine (Y401C). Both mutants were resistant to tetrodotoxin and saxitoxin. Whereas Na_v1.4-Y401C displayed an increased sensitivity to cadmium and extracellular QX-314, the serine substitution did not alter the sensitivity of Na_v1.4 to cadmium or QX-314. Our data indicates that while the serine residue determines the sensitivity of I_TTXr-Slow and I_TTXr-Per to tetrodotoxin and saxitoxin, it does not determine their insensitivity to QX-314 or their differential sensitivities to cadmium.This work was supported in part by grants from the Medical Research Service and the Rehabilitation Research Service, Department of Veterans Affairs, and by grants from The Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America.     

Molecular basis for pharmacological differences between brain and cardiac sodium channels

Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn²⁺ and Cd²⁺. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn²⁺ and Cd²⁺, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn²⁺ or Cd²⁺, but this mutation markedly reduces sensitivity to block by Ca²⁺ and Co²⁺. The double mutant channel (F385CN388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.     

Blockade of epithelial Na+ channels by triamterenes — Underlying mechanisms and molecular basis

The three subunits (α, β, γ) encoding for the rat epithelial Na⁺ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na⁺ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At −90 mV and −40 mV, the IC₅₀ values were 5 μM and 10 μM, respectively. The blockage by triamterene, which is a weak base with a pK _a of 6.2, was dependent on the extracellular pH. The IC₅₀ was 1 μM at pH 6.5 and only 17 μM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at −90 mV were 0.74 μM for the charged and 100.6 μM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC₅₀ values in the range of 0.1–20 μM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC α-subunit-deletion mutant, Δ278–283, which lacks a putative amiloride-binding site, induced a Na⁺ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms. Received: 2 January 1996/Accepted: 17 May 1996     

Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K+ channels

A swelling-activated, background K⁺ current in the corneal epithelium is characteristically activated by fenamates and inhibited by diltiazem. Fatty acids also stimulate this current, indicating that its origin is a lipid-sensitive mechano-gated 2P domain K⁺ channel. In the present study, modulation of TREK-1, TREK-2, and TRAAK channels by fenamates and diltiazem was examined. TREK-1, TREK-2, and TRAAK currents transiently expressed in COS-7 cells were recorded by the perforated-patch configuration. As previously reported, arachidonic acid (20 M) stimulated all of these channels, and a volatile anesthetic, halothane (1 mM) augmented TREK-1 and TREK-2 but not TRAAK. Flufenamic acid (FA, 100 M), niflumic acid (NA, 100 M), and mefenamic acid (MA, 100 M) markedly stimulated TREK-1, TREK-2, and TRAAK. The potency sequence for the activation of TREK-1 and TREK-2 was FA > NA = MA, and the potency sequence for the activation of TRAAK was FA = NA > MA. Diltiazem (1 mM) inhibited TREK-1 and TREK-2, but not TRAAK. In conclusion, fenamates are openers of the lipid-sensitive mechano-gated 2P domain K⁺ channels, and diltiazem may be a specific blocker for TREK. These novel findings could help to further understand channel functions of the mechano-gated 2P domain K⁺ channels.     

Sodium conductance in calcium channels of guinea-pig ventricular cells induced by removal of external calcium ions

An inward current characterized by a slow inactivation, was induced when the extracellular Ca^2– concentration was reduced by EGTA. It was suppressed by replacing external Na^– with Tris⁺ or by D-600, increased by epinephrine, and was not affected by TTX. These findings suggest that this current is carried by Na⁺ ions through the Ca channels. The Na current decreased in amplitude as the concentration of external divalent cations was elevated. Blocking the Na current by divalent cations could be approximated by a bimolecular interaction between divalent cation and channel, with a dissociation constant of 1.2 M for Ca²⁺ and 60 M for Mg²⁺. Single channel currents were recorded in the cell-attached configuration. With a pipette solution of pCa=7.5 or pCa>8, the single channel I-V relationship was linear and the slope conductance was 70–75 pS. For 40 mV depolarizations from the resting potential, unitary currents were smaller at pCa=6 than at pCa=7.5. However, single channel events, which were observed after the repolarizing step to the resting potential, were much the same amplitude. The open time histogram was fitted with a single exponential having a time constant of 1.9 ms at around –40 mV (pCa>8, with 5 M Bay K 8644 in the bath solution), which was decreased with increasing the Ca²⁺ concentration in the pipette solution. Noise power spectra of patch currents at pCa=6 revealed a high-frequency component at around 1500 Hz. These results suggest that Ca binding to the sites with a high affinity for Ca²⁺ blocks the Na conductance in Ca channels. Reduction of the unitary current at higher concentrations of Ca²⁺ might be attributed to a rapid block by Ca²⁺.     

Slow inactivation of muscle μ1 Na+ channels in permanently transfected mammalian cells

The slow inactivation of cloned muscle α-subunit Na⁺ channels was investigated using a Chinese hamster ovary cell line permanently transfected with rat muscle μ1 cDNA. Expression of μ1 Na⁺ channels was found in cells maintained for more than 6 months after transfection; > 70% of cells expressed ≥ 3 nA of Na⁺ current at +30 mV under whole-cell patch-clamp conditions. As expected, Na⁺ currents in these cells were blocked by tetrodotoxin as well as by μ-conotoxin. After prolonged depolarization (10 s at +30 mV) to inactivate voltage-gated Na⁺ channels, Na⁺ currents slowly reappeared over a time course of several minutes, during which time the cell was repolarized to the holding potential of −100 mV. This recovery from slow inactivation was best fitted by a double exponential function with τ₁ = 2.5 s (amplitude = 53%) and τ₂ = 83.4 s (amplitude = 38%). In contrast, the development of slow inactivation at +30 mV was best fitted by a single exponential function, with τ = 3.0 s. Steady-state slow inactivation (s _∞) had a midpoint potential (s ₀.₅) of −52 mV and a slope factor (k) of 7.8 mV. Elimination of fast inactivation by treatment with chloramine-T accelerated the development of slow inactivation significantly (by ≈four fold) but had little effect on recovery or on steady-state slow inactivation. Finally, as in cloned brain NaIIA Na⁺ channels, batrachotoxin abolished both fast and slow inactivation of μ1 Na⁺ channels. These results together suggest that slow inactivation takes place in the α-subunit of μ1 muscle Na⁺ channels and is governed by a μl protein region different from that governing fast inactivation. Received: 15 November 1995/Received after revision and accepted: 2 April 1996     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Sodium transport deficiency and sodium balance in gene‐targeted mice

Animals with induced or natural null mutations in renal NaCl and water transporter genes provide a powerful tool to study the physiological mechanisms that enable the kidney to optimize the match between glomerular filtration rate and tubular reabsorption. Deficiencies in the Na/H exchanger NHE3 and in the water channel aquaporin 1 (AQP1) cause reductions in proximal fluid absorption which are accompanied by proportionate decrements in glomerular filtration rate (GFR). Compensation of the transport defect by a reduction in filtered load is so efficient that clinically symptomatic Na losses are not observed in either NHE3 or AQP1 deficient animals. On the other hand, severe syndromes of salt wasting are caused by loss of function of the Na,K,2Cl‐cotransporter (NKCC2) in the thick ascending limb, or of the epithelial Na channel (ENaC) the collecting duct indicating that the severity of Na dysregulation is unrelated to the basal absorption of NaCl in a given nephron segment. In these states, the increased delivery of Na to downstream segments is not monitored by a sensor linked to the site of filtrate formation. In the absence of adaptations in the filtered load intrarenal compensation of a circumscribed NaCl malabsorption by adjustment of NaCl transport in other nephron segments is sometimes insufficient, particularly in the immature kidney of the newborn.     

Comparative histochemical and immunohistochemical study on xanthine oxidoreductase/xanthine oxidase in mammalian corneal epithelium

We have previously found that xanthine oxidase (one form of xanthine oxidoreductase that generates reactive oxygen species, such as superoxide radicals and hydrogen peroxide) is present in corneal epithelium of normal rabbit eye. It was suggested that the reactive oxygen species contribute to additional eye damage related to prolonged continuous contact lens wear and irradiation of the eye with UV-B light. To further explore the potential danger of xanthine oxidase as a source of reactive oxygen species, we have examined in the present paper whether xanthine oxidoreductase and xanthine oxidase are present in corneal epithelium of other mammalian species, employing immunohistochemical and enzyme histochemical methods. In corneal epithelium of normal eyes of ox, pig, guinea-pig, and rat xanthine oxidoreductase activity was detected by the tetrazolium salt reduction method and xanthine oxidase activity was localized by a method based on cerium ions capturing hydrogen peroxide. For the immunohistochemical demonstration of the enzymes, rabbit anti-bovine xanthine oxidase antibody, rabbit anti-human xanthine oxidase antibody and monoclonal mouse anti-human xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody were used. The immunohistochemical and enzyme histochemical results show that xanthine oxidoreductase and xanthine oxidase are present both as proteins and as active enzymes in the corneal epithelium of all animals studied. It is hypothesized that under various pathological states, xanthine oxidase-generated reactive oxygen species might contribute to eye damage.     

Chloride-selective channels of large conductance in bovine aortic endothelial cells

Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above ± 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or andenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.     

Upregulation of colonic ion channels in APC Min/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in almost all human colonic cancers. Disturbances in Na⁺ absorption have been observed in colonic cancer, and ion channels such as ether a go-go (Eag) or Ca²⁺-sensitive BK channels have been recognized for their oncogenic potential. APC ^Min/+ mice have reduced APC expression and develop multiple intestinal neoplasias (Min). Ion channels in the colonic epithelium were examined using electrophysiology and molecular techniques. APC ^Min/+ mice developed intestinal neoplasia and experienced a significant weight loss. Due to intestinal bleedings, the hematocrit was largely reduced and plasma aldosterone levels were enhanced. Rectal potential measurements in vivo indicated an increase in amiloride-sensitive Na⁺ absorption in APC ^Min/+ mice. Quantitative Ussing chamber studies demonstrated enhanced Na⁺ absorption via epithelial Na⁺ channels (ENaC) and suggested enhanced activity of oncogenic BK and Eag-1 channels. Patch clamp and fluorescence measurements on isolated crypts suggested enhanced K⁺ channel activity in the surface epithelium. ENaC-mRNA and membrane protein expression was enhanced in colonic surface epithelial cells. The data suggest that reduced expression of the APC gene with upregulation of the downstream proteins Akt and mTOR and subsequent hyperaldosteronism is paralleled by upregulation of oncogenic potassium channels and enhanced colonic Na⁺ absorption. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.