Characteristics Of Ion Channels Research Articles

Two channels, one program

The biophysical and structural characteristics of chloride ion channels have been studied extensively, but little is known about the molecular regulation and signaling pathways associated with these highly conserved proteins. Rutledge et al. (page 435) analyzed the activation of the CLH-3 chloride ion channel in C. elegans during meioitic cell cycle progression and in response to oocyte swelling. The work demonstrates the utility of the worm system in studying these channels, and suggests that CLH-3 and its putative mammalian orthologue, ClC-2, respond to similar regulatory inputs to carry out similar physiological functions. Figure A phosphatase inhibitor (CA) prevents meiotic maturation–induced current. The authors found that during oocyte maturation, or in response to oocyte swelling, CLH-3 is activated by serine/threonine dephosphorylation. RNAi inhibition demonstrates that the dephosphorylation is mediated by CeGLC-7α and CeGLC-7β, phosphatases that help to regulate meiotic and mitotic cell cycles in worms. Rat ClC-2 heterologously expressed in mammalian cells is also activated by serine/threonine dephosphorylation, suggesting that the two channels share a common regulatory mechanism, despite their wide evolutionary separation. Rutledge et al. suggest that both channels may depolarize membranes to transduce signals between cell types, such as worm oocytes and the surrounding contractile sheath cells, that are coupled by gap junctions. ▪

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue α helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/ V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as—no voltage dependence, only one unitary conductance, linear relation ship current–voltage—, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.

Voltage-gated K(+) channel activity in human prostate cancer cell lines of markedly different metastatic potential: distinguishing characteristics of PC-3 and LNCaP cells.

Although ion channels are known to contribute to a variety of basic cellular behaviors involved in the metastatic cascade, their role in metastasis per se has only recently been questioned. The hypothesis tested was whether K(+) channels were different between strongly metastatic PC-3 and weakly metastatic LNCaP human prostate cancer cell lines. The whole-cell configuration of the patch clamp recording technique was used to record voltage-gated currents from LNCaP and PC-3 cell lines. The responses to different voltage-clamp protocols, sensitivity to external Ca(2+), and addition of drugs and toxins were explored. Voltage-gated K(+) current density was significantly larger in LNCaP than PC-3 cells. In addition, the K(+) currents in a sub-population of PC-3 cells were Ca(2+)-sensitive. These properties reflected the differential metastatic character of the cells, the PC-3 cells appearing potentially more "excitable". Prostate cancer cells of varying metastatic ability can be distinguished by their ion channel characteristics. The possible contribution(s) of K(+) channel activity to development of malignancy needs exploration.

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Network stability from activity-dependent regulation of neuronal conductances.

Activity-dependent plasticity appears to play an important role in the modification of neurons and neural circuits that occurs during development and learning. Plasticity is also essential for the maintenance of stable patterns of activity in the face of variable environmental and internal conditions. Previous theoretical and experimental results suggest that neurons stabilize their activity by altering the number or characteristics of ion channels to regulate their intrinsic electrical properties. We present both experimental and modeling evidence to show that activity-dependent regulation of conductances, operating at the level of individual neurons, can also stabilize network activity. These results indicate that the stomatogastric ganglion of the crab can generate a characteristic rhythmic pattern of activity in two fundamentally different modes of operation. In one mode, the rhythm is strictly conditional on the presence of neuromodulatory afferents from adjacent ganglia. In the other, it is independent of neuromodulatory input but relies on newly developed intrinsic properties of the component neurons.

Regulation of ion channel expression in neural cells by hormones and growth factors.

Voltage-and ligand-gated ion channels are key players in synaptic transmission and neuron-glia communication in the nervous system. Expression of these proteins can be regulated at several levels (transcriptional, translational, or posttranslational) and by multiple extracellular factors in the developing and mature nervous system. A wide variety of hormones and growth factors have been identified as important in neural cell differentiation, which is a complex process involving the acquisition of cell-type-specific ion channel phenotypes. Much literature has already accumulated describing the structural and functional characteristics of ion channels, but relatively little is known about the factors that influence their synthesis and cell surface expression, although this area has generated considerable interest in the context of neural cell development. This article reviews several examples of regulated expression of these channels by cellular factors, namely peptide growth factors and steroid hormones, and discusses, where applicable, current understanding of molecular mechanisms underlying such regulation of voltage-and neurotransmitter-gated ion channels.

Molecular genetics of inherited long QT syndromes.

Unravelling the molecular cause of QT prolongation is one of the most exciting contributions of molecular biology to clinical cardiology in the last decade. Not only have mutations in four different genes been de-scribed, at least two additional chromosomal loci have been identified harbouring genes with an as yet unknown DNA sequence and protein function. Simultaneously cellular electrophysiology has provided the tools to study the effect of single base mutations on protein function. The development of molecular genetics in concert with molecular electrophysiology has provided substantial proof of the causative rela-tionship among DNA mutation, amino acid change and changes in ion channel characteristics. The physiological in vivo experiments in transgenic or gene targeted animals, showing prolonged repolarization and potential arrhythmias, will complete the evidence in the near future.

Pressure-activated cation channel in intact rat endocardial endothelium.

The endocardial endothelium (EE) regulates myocardial performance in response to humoral and mechanical stimuli. In vascular endothelium mechanosensitive ion channels (MSC) act as mechanosensors for hemodynamic changes. In the present study we examined whether MSC are present in intact EE of rat papillary muscle segments and characteristics of MSC are altered in experimental hypertension. MSC were investigated by the use of standard patch-clamp technique. For a comparative study, ion channel characteristics were determined in EE of two-kidney-one-clip rats and sham-operated controls. We identified a new class of MSC with a mean conductance of 21.8 +/- 4.4 (s.d.) pS for K+ and Na+ and of 4.1 +/- 1.5 pS for Ca2+. Channel activity was initiated by positive pipette pressure and blocked by negative pipette pressure. Channel open probability (Po) was characterized by its pressure sensitivity. Po increased from 0.06 at 10 mmHg to 0.37 and 0.55 at 20 mmHg and 30 mmHg, respectively. Gadolinium (20 microM), a blocker of MSC, completely inhibited channel activity. In some experiments activation of this pressure-activated channel (PAC) was followed by the opening of a Ca2(+)-dependent non-selective cation channel (NSC). This indicates that Ca2+ influx through PAC may be sufficient to increase intracellular Ca2+ concentration and thereby to activate neighboring NSC. In renovascular hypertension (2K1C), channel density of PAC was significantly increased compared to sham-operated controls. Channel density of NSC was not changed in 2K1C compared to sham-operated controls. A novel type of Ca2+ permeable MSC in intact EE of rat ventricular papillary muscle was identified, which is regulated by membrane pressure. PAC might be implicated in EE mechanotransduction by inducing an intracellular Ca2+ signal. Up-regulation of PAC density in EE from 2K1C might contribute to an altered mechanotransduction in hypertension.

Alamethicin-like behaviour of new 18-residue peptaibols, trichorzins PA. Role of the C-terminal amino-alcohol in the ion channel forming activity

The influences of peptide length, absence of a Glx (Gln/Glu) residue and the C-terminal amino alcohol on liposome permeabilization and ion-channel characteristics in planar lipid bilayers were examined with two 18-residue peptaibols, PA V and PA IX. As compared to the 20-residue alamethicin, both peptides belonging to the newly isolated trichorzin family, lack a proline in the N-terminal part and one of the two Gln/Glu residues in the C-terminal part of the sequence. The two analogues studied here differ among themselves in their C-terminal amino alcohol (tryptophanol for PA V and phenylalaninol for PA IX). These α-helical peptaibols modify to a similar extent the permeability of liposomes, as measured by leakage of a previously entrapped fluorescent probe. Monitoring tryptophanol fluorescence, a greater embedment of the peptide PA V is observed in cholesterol-free bilayers. Macroscopic conductance studies for PA V and PA IX display alamethicin-like current–voltage curves, with a similar voltage dependence, but a smaller mean number of monomers per conducting aggregate is estimated for the tryptophanol analogue, PA V. Single-channel recordings indicate faster current fluctuations for PA IX, while amplitude histograms show lower conductance levels for PA V. Apart from underlining the role of the mismatch between helix length and bilayer hydrophobic thickness, these results stress that the C-terminal tryptophanol favours a stabilization of the conducting aggregates.

Ion Channels in Epilepsy

There are specific alterations in the structure or function of ion channels in the epileptic brain. Some of these alterations may promote hyperexcitability, whereas others may protect neurons from the deleterious effects of epileptic discharges. With the use of human tissue resected from epilepsy patients and the comparison of cellular properties to those found in well-defined experimental models, we will continue to gain insight into the specific ion channel changes associated with epilepsies. Further genetic studies will help to elucidate the altered molecular mechanisms underlying ion channel changes in this devastating neurological disorder (Noebels, 1996). Whether it is a change in structure, function, or both, the study of ion channels in epilepsies will soon reveal specific characteristics of ion channels found only in epileptic tissue. If the altered properties of such ion channels cannot be found in control (nonepileptic) neurons, these channels might be called "epileptic" ion channels. An understanding of the specific structure, function, and pharmacology of these "epileptic" channels will yield important clues for future therapeutical approaches aimed at preventing epileptogenesis, and insight into the processes whereby ion channels become "epileptic" may finally open the way to prophylactic treatments of the epilepsies.

Colonic epithelium-enriched protein A4 is a proteolipid that exhibits ion channel characteristics.

Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.

Vagaries of artificial bilayers and gating modes of the SCl channel from the sarcoplasmic reticulum of skeletal muscle

The elucidation of the structure-function relation of reconstituted ion channel proteins into artificial planar bilayers is expected to be greatly influenced by the physical properties of these bilayers. The dependence of the gating mode of a voltage- and calcium-dependent small chloride channel (SCl channel) on the bilayer properties, as deduced from the specific bilayer capacitance, C b, was investigated. In bilayers having a C b value of 0.42 μF/cm 2 the SCl channel was in the “burst mode”. At a C b value of 0.28 μF/cm 2 the “burst mode” gave way to a “flickering mode”. At C b values between 0.24 and 0.28 μF/cm 2 channel bursts were often infrequent and brief. No ion channel activities were observed at C b values lower than 0.24 μF/cm 2. These results suggest that changes in bilayer thickness, mediated via n-decane, contribute to changes in channel gating mode, i.e. “gear shift”. Standardizing C b and its time-independence are important in comparative and detailed investigations of ion channel characteristics.

Regulation of potassium channels by protein kinases

Studies of the role of protein phosphorylation in the modulation of neuronal excitability are beginning to identify specific sites on ion channels that are substrates for serine/threonine kinases and that contribute to short-term and long-term regulation of current amplitude and kinetics. In addition, it is becoming apparent that phosphorylation of tyrosine residues may produce acute changes in the characteristics of ion channels. These recent findings are best illustrated by examining the Shaker superfamily of potassium channels.

Functional aspects of dexamethasone upregulated nicotinic acetylcholine receptors in C2C12 myotubes

Three days of treatment with the glucocorticoid dexamethasone (1 nM-1 microM) induced a concentration-dependent up-regulation of muscle nicotinic acetylcholine receptor (nAChR) in C2C12 mouse myotubes (EC50 = 10 +/- 7.3 nM), as assessed by [3H]alpha-BuTx binding. The maximum increase in binding amounted 148 +/- 17.6% of control. Parallel electrophysiological measurements employed the patch-clamp technique in cell-attached configuration. The nAChR single channel properties were investigated in the presence of carbachol (1 microM) in the pipette. Treatment with dexamethasone (1 microM, 1-5 days) induced an increase in the number of patches showing channel activity from 30 to 70%. Ion channel characteristics did not differ significantly in control and dexamethasone treated myotubes. Conductance was 32 +/- 3 vs 31 +/- 2 pS, respectively. The time constants of open time events tau 1 and tau 2 were 0.6 +/- 0.1 and 6.6 +/- 1 ms vs 0.6 +/- 0.1 and 6.6 +/- 1 ms, respectively. Closed duration's tau 1 and tau 2 were 1.1 +/- 0.2 and 110 +/- 12 ms vs 1.2 +/- 0.3 and 107 +/- 18 ms. In conclusion, dexamethasone upregulated nAChRs are functional and their electrophysiological parameters are similar to those found in control myotubes.

A generalized neuronal activation function derived from ion-channel characteristics

An abstract model neuron, simple enough to be used in analytical studies of neural networks, is defined. It incorporates neuronal adaptation, i.e. the coupling between neuronal activity and excitability, which can control the complexity of neural network dynamics. A generalized neuronal activation function is defined as the output activity, i.e. The firing rate of action potentials, as a function of an input and an excitability variable. For a biological neuron, the intracellular concentration of Ca ions is shown to be a useful excitability variable. The principal features of the generalized activation function are derived by means of a multicompartment model neuron with ionic currents described by Hodgkin-Huxley-type equations. As a relevant neuron type for investigations of cortical associative memory, a neocortical pyramidal cell of the regularly-spiking type is selected. An approximate analytical consideration suggests a simple form of independent variable of the generalized activation function. The complete numerical treatment verifies this form and displays a threshold character of the activation function. The derivation thereby pmvides an interpretation of dynamic threshold models of neuronal adaptation in terms of ionic mechanism. The threshold with respect to the input has well-defined dynamics directly given by the intracellular Ca dynamics. A comparison of the response of the Hodgkin-Huxley-type and reduced-model neumns to a varying input variable indicates that the reduced model is a usefui approximation even for the detailed behaviour of the firing rate. The generalized activation function also accounts for the dependence on neuromodnlation via an adaptability parameter. An absmt model neural network of units thus defined has the capacity to describe complex network dynamics and the simplicity to allow elucidation of mechanisms and applications to large-scale systems.

A generalized neuronal activation function derived from ion-channel characteristics

An abstract model neuron, simple enough to be used in analytical studies of neural networks, is defined. It incorporates neuronal adaptation, i.e. the coupling between neuronal activity and excitability, which can control the complexity of neural network dynamics. A generalized neuronal activation function is defined as the output activity, i.e. The firing rate of action potentials, as a function of an input and an excitability variable. For a biological neuron, the intracellular concentration of Ca ions is shown to be a useful excitability variable. The principal features of the generalized activation function are derived by means of a multicompartment model neuron with ionic currents described by Hodgkin-Huxley-type equations. As a relevant neuron type for investigations of cortical associative memory, a neocortical pyramidal cell of the regularly-spiking type is selected. An approximate analytical consideration suggests a simple form of independent variable of the generalized activation function. The c...

Membrane ionic currents and properties of freshly dissociated rat brainstem neurons.

It is well known that neuronal firing properties are determined by synaptic inputs and inherent membrane functions such as specific ionic currents. To characterize the ionic currents of brainstem cardio-respiratory neurons, cells from the hypoglossal (XII) nucleus and the dorsal motor nucleus of the vagus (DMX) were freshly dissociated and membrane ionic currents were studied under whole-cell voltage and current clamp. Both of these neurons showed a TTX-sensitive Na+ current with a much larger current density in XII than DMX neurons. This Na+ current had two (fast and slow) distinct inactivation decay components. The ratio of the magnitudes of the fast to slow component was roughly two-fold greater in DMX than in XII cells. Both DMX and XII neurons also showed a high voltage-activated Ca2+ current, but this current density was significantly greater (three-fold) in DMX than XII neurons. A relatively small amount of low-voltage activated Ca2+ current was also observed in DMX neurons, but not in the majority of XII cells. A transient and a sustained outward current components were observed in DMX cells, but only sustained currents were present in XII neurons. These outward currents had a reversal potential of about -70 mV with 3 mM external K+ and -30 mV with 25 mM K+, and substitution of K+ with cesium and tetraethylammonium suppressed more than 90% the outward currents, indicating that most outward currents were carried by K+. The transient outward current consisted of two components with one sensitive to 4-aminopyridine and the other to intracellular Ca2+. In XII neurons, BRL 38227 (lemakalim), an ATP-sensitive K+ (KATP) channel activator, increased the sustained K+ currents by 10% of control, and glibenclamide, a KATP channel blocker, decreased the sustained K+ currents by 20%. Evidence for the presence of an inward rectifier K+ current was also obtained from both XII and DMX neurons. These results on XII and DMX neurons indicate that (1) the methods used to dissociate neurons provide a useful means to overcome voltage clamp technical difficulties; (2) ion channel characteristics such as density and biophysical properties of DMX neurons are very different from those of XII neurons; and (3) several newly discovered membrane ionic currents are present in these cells.

Patch clamp studies of human placental cytotrophoblast cells in culture

Despite the importance of the trophoblast in the exchange of ions across the placenta, little is known of the transport mechanisms for ions in trophoblast cells. We have used patch clamp techniques to study ion channels in human cytotrophoblast cells in culture (days 2–8).In an initial survey, 11 cell-attached and 13 inside-out patches were studied and 13 channels were observed. Of these, the following four channels were identified: a) a large conductance Cl − channel (386±20 pS; n=3) with characteristics typical of maxi Cl − channels (Gogelein, 1988) and previously identified at the microvillous membrane of the human placental syncytiotrophoblast at term (Brown et al., 1993), b) a smaller conductance channel (19.5±1.0 pS; n=3) present in inside-out patches which may be Cl − selective, c) a Ca 2+ activated K + channel with a maximum conductance of 202 pS in cell attached and 182 pS in inside-out patches, and d) a 56 pS channel (maximum conductance at negative potentials of 56±3 pS; n=4) present in cell attached patches which may be selective for K + . The ion selectivities, kinetics, pharmacology, and regulation of the channelshave yet to be studied and their roles in trophoblast transport are unknown at present. Human cytotrophoblast cells in culture provide an ideal system to study the incidence and characteristics of ion channels and how these may change throughout trophoblast differentiation.

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K + ion channels (2–6 pA) using the patch-clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K + flux is due solely to concentration differences. Although conductances are in the range expected for K + channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.

A depolarization can trigger Ca 2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca 2+ ([Ca 2+] i) and intracellular pH (pH i), a K +-dependent transient hyperpolarization which may involve K + channels, and a depolarization which depends on external Ca 2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K +-free seawater raised the pH i, caused a small increase in 45Ca 2+ uptake, and triggered some AR. When the cells were depolariźed with KCl (30 m M) 40–60 sec after the induced hyperpolarization, the pH i decreased and there was a significant increase in 45Ca 2+ uptake, [Ca 2+] i, and the AR. This waiting time was necessary in order to allow the pH i change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pH i, may regulate Ca 2+ transport systems involved in this process. Because of the key role played by K + in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.