Na+ channels and amiloride-induced noise in the mammalian colon epithelium

(1) The effects of the Na+-channel blocker, amiloride, on the short-circuit current carried by Na+ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na+-current noise showed a Lorentzian component. When the Na+ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Macroscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na+ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na+-channel density were calculated at room temperature and at 37 degrees C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na+ channel in the colon were similar to those found for Na+ channels in other tight epithelia.     

Propagation of photon noise and information transfer in visual motion detection

The extraction of the direction of motion from the time varying retinal images is one of the most basic tasks any visual system is confronted with. However, retinal images are severely corrupted by photon noise, in particular at low light levels, thus limiting the performance of motion detection mechanisms of what sort so ever. Here, we study how photon noise propagates through an array of Reichardt-type motion detectors that are commonly believed to underlie fly motion vision. We provide closed-form analytical expressions of the signal and noise spectra at the output of such a motion detector array. We find that Reichardt detectors reveal favorable noise suppression in the frequency range where most of the signal power resides. Most notably, due to inherent adaptive properties, the transmitted information about stimulus velocity remains nearly constant over a large range of velocity entropies. Action editor: Matthew Wiener     

Structure and function of amiloride-sensitive Na+ channels

A new molecular biological epoch in amiloride-sensitive Na⁺ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na⁺ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na⁺ channel dysfunction has been implicated [17, 62].Epithelia Na⁺ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na⁺ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers.     

Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels

Epithelial Na(+) channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na(+). The down-regulation of ENaC activity by external Na(+) is referred to as Na(+) self-inhibition. We investigated the structural determinants of Na(+) self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na(+) currents following a rapid increase of bath Na(+) concentration. Our results indicated that wild-type mouse alphabetagammaENaC has intrinsic Na(+) self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His(239) (gammaH239R, gammaH239D, and gammaH239C) in the extracellular loop of the gammaENaC subunit prevented Na(+) self-inhibition whereas mutations of the corresponding His(282) in alphaENaC (alphaH282D, alphaH282R, alphaH282W, and alphaH282C) significantly enhanced Na(+) self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na(+) self-inhibition.     

Point mutations in alpha-subunit of human cardiac Na+ channels alter Na+ current kinetics

Dietary polyunsaturated fatty acids (PUFAs) prevent ischemia-induced fatal cardiac arrhythmias in animals and probably in humans. This action results from inhibition of ion currents for Na+, Ca2+, and possibly other ions. To extend understanding of this protection we are seeking a possible binding site for the PUFAs on the alpha-subunit of the human cardiac Na+ channel, hH1alpha, transiently expressed in HEK293t cells. Three mutated single amino acid substitutions with lysine were made in the alpha-subunit at Domain 4-Segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406. These are in the putative sites of binding of local anesthetics and batrachotoxin, respectively. The mutants F1760K, Y1767K, and N406K, separately and to different extents, affected the current density, the steady-state inactivation potential, accelerated inactivation, delayed recovery from inactivation, and affected voltage-dependent block, but did not affect activation of the hH1alpha. It is essential to learn that single point mutations in D1-S6 and D4-S6 alone significantly modify the kinetics of human cardiac hH1alpha Na+ currents. The effects of PUFAs on these mutant channels will be the subject of subsequent reports.     

Voltage-sensitive Na+ channels: motifs, modes and modulation.

Much recent progress has been made in understanding the structural organization and functional properties of voltage-dependent Na+ channels, in particular in the areas of activation, ion conductance, and inactivation. At the same time, however, electrophysiological studies have revealed new, more complex functional properties in the form of at least two gating modes and the existence of as yet unidentified modulatory factors.     

Roles of glucose in photoreceptor survival

Vertebrate photoreceptor neurons have a high demand for metabolic energy, and their viability is very sensitive to genetic and environmental perturbations. We investigated the relationship between energy metabolism and cell death by evaluating the metabolic effects of glucose deprivation on mouse photoreceptors. Oxygen consumption, lactate production, ATP, NADH/NAD(+), TCA cycle intermediates, morphological changes, autophagy, and viability were evaluated. We compared retinas incubated with glucose to retinas deprived of glucose or retinas treated with a mixture of mitochondrion-specific fuels. Rapid and slow phases of cell death were identified. The rapid phase is linked to reduced mitochondrial activity, and the slower phase reflects a need for substrates for cell maintenance and repair.     

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Predominance of poorly reopening single Na+ channels and lack of slow Na+ inactivation in neonatal cardiocytes

Summary Elementary Na⁺ currents through single cardiac Na⁺ channels were recorded at –50 mV in cell-attached patches from neonatal rat cardiocytes kept at holding potentials between –100 and –120 mV.Na⁺ channel activity may occur as burst-like, closely-timed repetitive openings with shut times close to 0.5–0.6 msec, indicating that an individual Na⁺ channel may reopen several times during step depolarization. A systematic quantiative analysis in 19 cell-attached patches showed that reopening may be quite differently pronounced. The majority, namely 16 patches, contained Na⁺ channels with a low tendency to reopen. This was evidenced from the average value for the mean number of openings per sequence, 2.5. Strikingly different results were obtained in a second group of three patches. Here, a mean number of openings per sequence of 3.42, 3.72, and 5.68 was found. Ensemble averages from the latter group of patches revealed macroscopic Na⁺ currents with a biexponential decay phase. Reconstructed Na⁺ currents from patches with poorly reopening Na⁺ channels were devoid of a slow decay component. This strongly suggests that reopening may be causally related to slow Na⁺ inactivation. Poorly pronounced reopening and, consequently, the lack of slow Na⁺ inactivation could be characteristic features of neonatal cardiac Na⁺ channels.     

Structural and functional organization of Na+ transport in epithelial systems. I. Epithelial Na+ channels

The review summarizes recent data on the structural and functional organization and regulation mechanisms of Na+ transport in epithelial systems. The review is focused on the structure, function, regulation and pathology of epithelial Na+ channels, which are critical for Na+ homeostasis maintenance and blood pressure control.     

Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.