Copper modulation of ion channels of PrP[106-126] mutant prion peptide fragments.

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.

Channel activity of deamidated isoforms of prion protein fragment 106-126 in planar lipid bilayers.

Using the lipid bilayer technique, we have found that age-related derivatives, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), of the prion protein fragment 106-126 (PrP[106-126] (Asn108)) form heterogeneous ion channels. The deamidated isoforms, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), showed no enhanced propensity to form heterogeneous channels compared with PrP[106-126] (Asn108). One of the PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels had three kinetic modes. The current-voltage (I-V) relationship of this channel, which had a reversal potential, E(rev), between -40 and -10 mV close to the equilibrium potential for K+ (E(K)-35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 62.5 pS at positive potentials between 0 and 140 mV. The probability (P(o)) and the frequency (F(o)) of the channel being open had inverted and bell-shaped curves, respectively, with a peak at membrane potential (V(m)) between -80 and +80 mV. The mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves. The biophysical properties of PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels and their response to Cu(2+) were similar to those of channels formed with PrP[106-126] (Asn108). Cu(2+) shifted the kinetics of the channel from being in the open state to a "burst state" in which rapid channel activities were separated by long durations of inactivity. The action of Cu(2+) on the open channel activity was both time-dependent and voltage-dependent. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in the conductance of the channel indicated that Cu(2+) binds at the mouth of the channel. Consistently with the hydrophilic and structural properties of PrP[106-126], the Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the Cu(2+) binding site could be located at M(109) and H(111) of this prion fragment.

Mechanisms of prion-induced modifications in membrane transport properties: implications for signal transduction and neurotoxicity.

Prion-related encephalopathies are associated with the conversion of a normal cellular isoform of prion protein (PrP(c)) to an abnormal pathologic scrapie isoform (PrP(Sc)). The conversion of this single polypeptide chain involves a reduction in the alpha-helices and an increase in beta-sheet content. This change in the content ratio of alpha-helices to beta-sheets may explain the diversity in the proposed mechanisms of action. Many of the pathogenic properties of PrP(Sc), such as neurotoxicity, proteinase-resistant properties and induction of hypertrophy and proliferation of astrocytes, have been attributed to the peptide fragment corresponding to residues 106-126 of prion (PrP[106-126]). In particular, the amyloidogenic and hydrophobic core AGAAAAGA has been implicated in modulation of neurotoxicity and the secondary structure of PrP[106-126]. Because of some similarities between the properties of PrP[106-126] and PrP(Sc), the former is used as a useful tool to characterize the pharmacological and biophysical properties of PrP(Sc) in general and of that domain in particular, by various laboratories. However, it is important to note that by no means can PrP[106-126] be considered a complete equivalent to PrP(Sc) in function. Several hypotheses have been proposed to explain prion-induced neurodegenerative diseases. These non-exclusive hypotheses include: (i) changes in the membrane microviscosity; (ii) changes in the intracellular Ca(2+) homeostasis; (iii) superoxide dismutase and Cu(2+) homeostasis; and (iv) changes in the immune system. The prion-induced modification in Ca(2+) homeostasis is the result of: (1) prion interaction with intrinsic ion transport proteins, e.g. L-type Ca(2+) channels in the surface membrane, and IP(3)-modulated Ca(2+) channels in the internal membranes, and/or (2) formation of cation channels. These two mechanisms of action lead to changes in Ca(2+) homeostasis that further augment the abnormal electrical activity and the distortion of signal transduction causing cell death. It is concluded that the hypothesis of the interaction of PrP[106-126] with membranes and formation of redox-sensitive and pH-modulated heterogeneous ion channels is consistent with: (a) PrP-induced changes in membrane fluidity and viscosity; (b) PrP-induced changes in Ca(2+) homeostasis (and does not exclude changes in endogenous Ca(2+) transport pathways and Cu(2+) homeostasis); (c) PrP role as an antioxidant; and (d) the PrP structural properties, i.e. beta sheets, protein aggregation, hydrophobicity, functional significance of specific amino acids (e.g. methionine, histidine) and regulation with low pH.

Mechanisms of amyloid beta protein-induced modification in ion transport systems: implications for neurodegenerative diseases.

1. Alzheimer's disease (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. Abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss. 2. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AbetaP[1-42] and AbetaP[1-43] and soluble nonpathologic AbetaP[1-40]. Both APP and AbetaP interact with ion transport systems. AbetaP induces a wide range of effects as the result of activating a cascade of mechanisms. 3. The major mechanisms proposed for AbetaP-induced cytotoxicity involve the loss of Ca2+ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca2+ homeostasis could be the result of (1) changes in endogenous ion transport systems, e.g. Ca2+ and K+ channels and Na+/K+-ATPase, and membrane receptor proteins, such as ligand-driven ion channels and G-protein-driven releases of second messengers, and (2) formation of heterogeneous ion channels. 4. The consequences of changes in Ca2+-homeostasis-induced generation of ROS are (a) direct modification of intrinsic ion transport systems and their regulatory mechanisms, and (b) indirect effects on ion transport systems via peroxidation of phospholipids in the membrane, inhibition of phosphorylation, and reduction of ATP levels and cytoplasmic pH. 5. We propose that in AD, AbetaP with its different conformations alters cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding plaque formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is not only central for AD but also may explain other malfunctioned protein-processing-related pathologies.

Diversity of amyloid beta protein fragment [1-40]-formed channels.

1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AbetaP) 1-40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AbetaP[1-40]-formed ion channels have been identified: (i) The AbetaP[1-40]-formed "bursting" fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV. 17 pS (250/50 mM KCl cis/trans) at -160 mV, and the nonlinear current-voltage relationship drawn to a third-order polynomial, (b) selectivity sequence PK > PNa > PLi = 1.0:0.60:0.47, (c) Po of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn2+-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AbetaP[1-40]-formed "spiky" fast cation channel was characterized by (a) a similar kinetics to the "bursting" fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at -160 mV, the current-voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence PRb > (iii) The AbetaP[1-40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at -160 mV and (b) inactivation at Vms more negative than -120 and more positive than +120 mV. (iv) The AbetaP[1-40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0-589 pS (in 250/50 mM KCI) at +140 mV and 0-704 pS (in 250/50 mM KCl) at -160 mV. (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at -76 mV, (c) the value of PK/Pcholine for the fast mode was 3.9 and selectivity sequence PK > PCs > PNa > PLi = 1.0:0.94:0.87:0.59. The value of PK/Pcholine for the slow mode was 2.7 and selectivity sequence PK > FNa > PLi > PCs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn2+-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn2+. 2. It has been suggested that, although the "bursting" fast channel, the "spiky" fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions. 3. Furthermore, the formation of beta-sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels.

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.

Protein aggregation and deposition: implications for ion channel formation and membrane damage.

Protein deposition, aggregation, and formation of amyloids are associated with a wide range of pathologies, including several neurodegenerative diseases. Aggregation and deposition are a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment. The mechanism of protein deposition and aggregation is triggered when the hydrophobic and positively charged regions of the misfolded proteins are exposed. The cells aim to regulate these misfolded and malfunctioning aggregation-prone proteins by degradation mechanisms, e.g., proteosomes, and/or by storing them in specialized compartments, e.g., Russell bodies and aggresomes. During these processes, however, some aggregation-prone protein intermediates are capable of aggregation and forming beta-sheet based channels in various negatively charged cellular membranes. Adverse cellular conditions, transitional metals, cellular proteins, and genetic mutations play an important role in the formation and function of these non-intrinsic channels. These channels, which can damage membrane function, are pathologic because they can disrupt the metabolic, ionic, and water homeostasis and distort signal transduction. We propose that different conformations of aggregation-prone proteins could alter cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding fibril formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is a general mechanism that may explain other malfunctioning protein processing-related pathologies.

Prion peptide fragment PrP[106-126] forms distinct cation channel types.

Using the lipid bilayer technique we have optimized the recording conditions and confirmed that PrP[106-126] (KTNMKHMAGAAAAGAVVGGLG) forms single ion channels. Based on the conductance and kinetic parameters of the single channel currents recorded in 250/50 mM KCl cis/trans we have found that the PrP[106-126]-formed heterogeneous cation channels that differ in their conductance and kinetic properties. The most frequently observed PrP[106-126]-formed single cation channels were those of: (a) a GSSH- and TEA-sensitive channel with fast kinetics (n = 47). The current-voltage (I-V) relationship of this channel, that has a reversal potential E(rev) of -33 mV close to the equilibrium potential for K(+) (E(K) -35 mV), exhibited inward and outward rectification. The values of the maximal slope conductance (g(max)) were 138 and 141 pS at positive and negative potentials, respectively. The values of the permeability ratios were 1.0:0.87:0.72:0.49:0.41 for K(+) > Rb(+) > Na(+) > Cs(+) > Li(+) respectively. The probability of the channel being open (P(o)) and the frequency (F(o)) had bell-shaped curves with a peak at membrane potential (V(m)) between -10 and -5 mV whereas the mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves; (b) a 4'-4'-dithiodipyridin (DTT)-sensitive channel with slow kinetics (n = 32). The I-V relationship of this channel that had an E(rev) of -35 mV and a g(max) of 41 pS at positive V(m) was non-linear. The parameter P(o) increased at positive V(m) to 0.6-0.7 at +80 mV. F(o) had an asymmetrical bell-shaped curve with a peak of 314 events/sec at -80 mV. The values of T(o) and T(c) were 312 and 164 msec at +120 mV, respectively; (c) a large channel (n = 24 channels) that had five equally spaced subconductances showed time-dependent fast and slow transitions at positive and negative V(m), respectively. The inactivation ratio I(ss)/I(i) was V(m) dependent and described by a bell-shape. The I-V relationship of this channel that had a E(rev) of -22 mV was non linear. The value of g(max) was 900 and 1444 pS at positive and negative V(m)s, respectively. The value of P(o) was 0.6 at negative V(m)s between -160 and -80 mV and 0.23 at +140 mV. F(o) increased from 22 events/sec at -160 mV to 80-100 events/sec at between +80 and +100 mV. T(o) decreased from 375 msec between -160 and -80 mV to 1-2 msec at V(m)s between 0 and +160 mV. In contrast, T(c) decreased from 160-240 msec at membrane voltages (V(m)s) between -160 and -80 mV. The biophysical properties of these channels indicate that they are capable of modifying cellular functions via modification of V(m) and electrolyte homeostasis of the cell.

Biophysical and molecular properties of annexin-formed channels.

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.

Properties of cytotoxic peptide-formed ion channels.

Cytotoxic peptides are relatively small cationic molecules such as those found 1) in venoms, e.g., melittin in bee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, and lycotoxin I and II in wolf spider; 2) in skin secretions (e.g., magainin I and II from Xenopus laevis, dermaseptin from frog, antimicrobials from carp) and cells of the immune system (e.g., insect, scorpion, and mammalian defensins and cryptdins); 3) as autocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic beta-cells, prion peptide fragment 106-126 [PrP-(106-126)], and amyloid beta-protein (AbetaP) cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of beta-sheets, e.g., mammalian defensins, AbetaP, amylin, and PrP-(106-126), whereas the larger cytotoxic peptides have several domains composed of both alpha-helices and beta-sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modify cell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These two nonexclusive mechanisms of action lead to changes in second messenger systems that further augment the abnormal electrical activity and distortion of the signal transduction causing cell death.

Temperatures in and around mandibular fractures.

This study measured the temperature in and around mandibular fractures in 20 anaesthetized patients. A fine calibrated thermocouple attached to a digital thermometer was used to measure temperature at the bone surface margin of a mandibular fracture and 5 and 10 mm from the fracture; within the fracture against the bone at 5, 7 and 10 mm depths and at the base of 7 mm deep biopsy cavities 5 and 10 mm distant from the fracture line immediately after biopsy, then 1 and 2 min later. On the surface the temperature was approximately 1 degrees C warmer than at the 5 and 10 mm sites distant from the fracture. Temperature reduced at the 1 and 2 min readings after the biopsy cavity cutting. This study confirmed that the surface temperature is lower than internal bone temperatures.

Hydrogen peroxide inhibits chloride channels of the sarcoplasmic reticulum of skeletal muscle.

Data obtained with the lipid bilayer technique indicate that cis (cytoplasmic) concentration of 4.4-22 mm hydrogen peroxide (H(2)O(2)), is a water-soluble oxidant. [H(2)O(2)](cis) (n = 26) reversibly inhibits the multisubconductance SCl channel of the sarcoplasmic reticulum vesicles from rabbit skeletal muscle. At -40 mV, the mean values of the current amplitude (I) and the probability of the SCl channel being open (P(o)) were reduced significantly (n = 8) from -6.14 +/- 0.42 pA and 0.69 +/- 0.06 (for all conductance levels) in control 0.0 mm [H(2)O(2)](cis) to -1.10 +/- 0.51 pA and 0. 13 +/- 0.04 (for the intermediate subconductance states) in 8.8 mm [H(2)O(2)](cis), respectively. The [H(2)O(2)](cis)-induced decrease in P(o) is mainly due to a decrease in the mean open time T(o). The mechanism of [H(2)O(2)](cis) effects on the multiconductance SCl channel is characterized by a mode shift in the channel state from the main conductance state to the low subconductance states. The estimated concentration of the [H(2)O(2)](cis) for the half inhibitory constant, K(i), was 11.78 mm, higher than the estimated 8. 0 and 8.1 mm for the parameters P(o) and T(o), respectively, indicating that the conductance of the SCl channel is less sensitive than the gating kinetics of the channel. After a lag period of between 30 to 60 sec, the lipophilic SH-oxidizing agent 4, 4'-dithiodipyridine (4,4'-DTDP) added to the cis side at 1.0 mm removed the inhibitory effects of 8.8 mm [H(2)O(2)](cis). The 4, 4'-DTDP-enhanced SCl channel activity was blocked after the addition of 0.5 mm ATP to the cis side of the channel. The addition of 1.0 mm 4,4'-DTDP to the cis or trans solutions facing an SCl channel already subjected to 0.5 mm [ATP](cis) or [ATP](trans) failed to activate the ATP-inhibited SCl channel. These findings suggest that 4,4'-DTDP is not preventing the binding of ATP to its binding site on the channel protein. The interaction of H(2)O(2) with the SCl channel proteins is consistent with a thiol-disulfide redox state model for regulating ion transport, where SH groups can directly modify the function of the channel and/or the availability of regulatory sites on the channel proteins. The H(2)O(2) effects on the Ca(2+) countercurrent through the SCl channel are also consistent with H(2)O(2)-modification of the mechanisms involved in the Ca(2+) regulation, which underlies excitation-contraction coupling in skeletal muscle.

A component of platypus (Ornithorhynchus anatinus) venom forms slow-kinetic cation channels.

The lipid bilayer technique is used to examine the biophysical properties of anion and cation channels frequently formed by platypus (Ornithorhynchus anatinus) venom (OaV). The OaV-formed anion channel in 250/50 mm KCl cis/trans has a maximum conductance of 857 +/- 23 pS (n = 5) in 250/50 mm KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent inactivation that can be removed by depolarizing voltage steps more positive than the reversal potential for chloride, E(Cl), (+40 mV). The reversal potential of the OaV-formed slow current activity in 250/50 mm KCl cis/trans is close to the potassium equilibrium potential (E(K)) of -40 mV. The conductance values for the slow channel are 22.5 +/- 2.6 pS and 41.38 +/- 4.2 pS in 250/50 and 750/50 mm cis/trans, respectively. The gating kinetics of the slow ion channels are voltage-dependent. The channel open probability (P(o)) is between 0.1 and 0.8 at potentials between 0 and +140 mV. The channel frequency (F(o)) increases with depolarizing voltages between 0 and +140 mV, whereas mean open time (T(o)) and mean closed time (T(c)) decrease. Ion substitution experiments of the cis solution show that the channel has conductance values of 21.47 +/- 2. 3 and 0.53 +/- 0.1 pS in 250 mm KCl and choline Cl, respectively. The amplitude of the single channel current is dependent on [K(+)](cis) and the current reversal potential (E(rev)) responds to increases in [K(+)](cis) by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K(+)](cis) can be best described by a third order polynomial fit. At +140 mV, the values of the maximal single channel conductance (gamma(max)) and the concentration for half maximal gamma (K(s)) are 38.6 pS and 380 mm and decline to 15.76 pS and 250 mm at 0 mV, respectively. The ion selectivity of the channel to K(+), Na(+), Cs(+) and choline(+) was determined in ion substitution experiments. The permeability values for P(K(+)):P(Na(+)):P(Cs(+)):P(choline(+)) were 1:1:0.63:0.089, respectively. On the other hand, the activity of the slow channel was eliminated (Fig. 7B). The slow channel was reversibly inhibited by [TEA(+)](trans) and the half-maximal inhibitory concentration (K(i)) was approximately 48 mm.

Calcium dependence of C-type natriuretic peptide-formed fast K(+) channel.

The lipid bilayer technique was used to characterize the Ca(2+) dependence of a fast K(+) channel formed by a synthetic 17-amino acid segment [OaCNP-39-(1-17)] of a 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchus anatinus) venom (OaV). The OaCNP-39-(1-17)-formed K(+) channel was reversibly dependent on 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic) Ca(2+) concentration ([Ca(2+)](cis)). The channel was fully active when [Ca(2+)](cis) was >10(-4) M and trans (luminal) Ca(2+) concentration was 1.0 mM, but not at low [Ca(2+)](cis). The open probability of single channels increased from zero at 1 x 10(-6) M cis Ca(2+) to 0.73 +/- 0.17 (n = 22) at 10(-3) M cis Ca(2+). Channel openings to the maximum conductance of 38 pS were rapidly and reversibly activated when [Ca(2+)](cis), but not trans Ca(2+) concentration (n = 5), was increased to >5 x 10(-4) M (n = 14). Channel openings to the submaximal conductance of 10.5 pS were dominant at >/=5 x 10(-4) M Ca(2+). K(+) channels did not open when cis Mg(2+) or Sr(2+) concentrations were increased from zero to 10(-3) M or when [Ca(2+)](cis) was maintained at 10(-6) M (n = 3 and 2). The Hill coefficient and the inhibition constant were 1 and 0.8 x 10(-4) M cis Ca(2+), respectively. This dependence of the channel on high [Ca(2+)](cis) suggests that it may become active under 1) physiological conditions where Ca(2+) levels are high, e.g., during cardiac and skeletal muscle contractions, and 2) pathological conditions that lead to a Ca(2+) overload, e.g., ischemic heart and muscle fatigue. The channel could modify a cascade of physiological functions that are dependent on the Ca(2+)-activated K(+) channels, e.g., vasodilation and salt secretion.

Characterization of a C-type natriuretic peptide (CNP-39)-formed cation-selective channel from platypus (Ornithorhynchus anatinus) venom.

1. The lipid bilayer technique is used to characterize the biophysical and pharmacological properties of a novel, fast, cation-selective channel formed by incorporating platypus (Ornithorhynchus anatinus) venom (OaV) into lipid membranes. 2. A synthetic C-type natriuretic peptide OaCNP-39, which is identical to that present in platypus venom, mimics the conductance, kinetics, selectivity and pharmacological properties of the OaV-formed fast cation-selective channel. The N-terminal fragment containing residues 1-17, i.e. OaCNP-39(1-17), induces the channel activity. 3. The current amplitude of the TEACl-insensitive fast cation-selective channel is dependent on cytoplasmic K+, [K+]cis. The increase in the current amplitude, as a function of increasing [K+]cis, is non-linear and can be described by the Michaelis-Menten equation. At +140 mV, the values of gammamax and KS are 63.1 pS and 169 mM, respectively, whereas at 0 mV the values of gammamax and KS are 21.1 pS and 307 mM, respectively. gammamax and KS are maximal single channel conductance and concentration for half-maximal gamma, respectively. The calculated permeability ratios, PK:PRb:PNa:PCs:PLi, were 1:0.76:0.21:0.09:0.03, respectively. 4. The probability of the fast channel being open, Po, increases from 0.15 at 0 mV to 0.75 at +140 mV. In contrast, the channel frequency, Fo, decreases from 400 to 180 events per second for voltages between 0 mV and +140. The mean open time, To, increases as the bilayer is made more positive, between 0 and +140 mV. The mean values of the voltage-dependent kinetic parameters, Po, Fo, To and mean closed time (Tc), are independent of [KCl]cis between 50 and 750 mM (P > 0. 05). 5. It is proposed that some of the symptoms of envenomation by platypus venom may be caused partly by changes in cellular functions mediated via the OaCNP-39-formed fast cation-selective channel, which affects signal transduction.

Synthetic mammalian C-type natriuretic peptide forms large cation channels.

We report the first evidence that synthetic human C-type natriuretic peptide-22 and the OaC-type natriuretic peptide-39(18-39), a 22 amino acid fragment of the OaC-type natriuretic peptide-39 from platypus venom, can function directly by forming a novel voltage-gated weakly cation-selective channel in negatively charged artificial lipid bilayer membranes. The channel activity is characterized by a tendency for inactivation at negative voltages, e.g. -60 and -70 mV, whereas at positive voltages the channel is fully open. The channel has a maximal cord conductance of 546+/-23 pS (n = 16) and shows weak outward rectification. The sequence and the permeability ratios were P(K)+: P(Cs)+: P(Na)+: P(choline)+ 1:0.88:0.76:0.13, respectively. The addition of 50 mM TEA+ cis (a blocker of outwardly rectifying K+ channels), 20 mM Cs+ cis (a blocker of inwardly rectifying K+ channels) or 0.5 mM glibenclamide cis (a blocker of ATP-sensitive K+ channels) to the cis chamber did not affect the conductance or the kinetics of the OaC-type natriuretic peptide-39(18-39)-formed channels (n = 2-5). It is concluded that the weak cation selectivity, large conductance and high open probability as well as their voltage dependency are consistent with the ability of these peptides to cause that loss of compartmentation of the membrane, which is a characteristic feature of adverse conditions that cause C-type natriuretic peptide-related pathologies.

Role of natriuretic peptides in ion transport mechanisms.

Natriuretic peptides (NP) act as ligands on the guanylyl cyclase family of receptors. The NP binding site on these receptors is extracellular and the guanylyl cyclase and protein kinase domains are intracellular. The guanylyl cyclase receptor catalyzes the synthesis of the second messenger molecule, cGMP, which activates protein kinase. This in turn is involved in the phosphorylation of various ion transport proteins. Ion transport proteins, which are modulated by NP and are thought to underlie the natriuretic and diuretic actions of NP, include: (a) calcium-activated K+ channels; (b) ATP-sensitive K+ channels; (c) inwardly-rectifying K+ channels; (d) outwardly-rectifying K+ channels; (e) L-type Ca2+ channels; (f) Cl- channels including cystic fibrosis transmembrane conductance regulator Cl- channels; (g) Na+- K+ 2Cl- co-transporter; (h) Na+- K+ ATPase; (i) Na+ channels; (j) stretch-activated channels; and (k) water channels. It appears that NP modulate the kinetics, rather than the conductance, of ion channels. Some of these channels, like the Ca2+, ATP-sensitive K+ and stretch-activated channels, are also involved in NP secretion. In addition, the structural properties of the NP, e.g., ovCNP-22 and ovCNP-39, appear to confer on them the ability to form ion channels. These CNP-formed ion channels can modify the trans-membrane signal transduction and second messenger systems underlying NP-induced pathological effects.

pH-modulation of chloride channels from the sarcoplasmic reticulum of skeletal muscle.

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pHcis) and luminal pH (pHtrans) was investigated using the lipid bilayer-vesicle fusion technique. Low pHcis 6.75-4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pHcis 7.26-7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65-75 pS) whereas at low pHcis 6.75-4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5-40 pS). Similarly, low pHtrans 4.07, but not pHtrans 6.28, modified the activity of SCl channels. The effects of low pHcis are pronounced at 10(-3) and 10(-4) M [Ca2+]cis but are not apparent at 10(-5) M [Ca2+]cis, where the subconductances of the channel are already prominent. Low pHcis-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pHcis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.

Interaction of reactive oxygen species with ion transport mechanisms.

The use of electrophysiological and molecular biology techniques has shed light on reactive oxygen species (ROS)-induced impairment of surface and internal membranes that control cellular signaling. These deleterious effects of ROS are due to their interaction with various ion transport proteins underlying the transmembrane signal transduction, namely, 1) ion channels, such as Ca2+ channels (including voltage-sensitive L-type Ca2+ currents, dihydropyridine receptor voltage sensors, ryanodine receptor Ca2+-release channels, and D-myo-inositol 1,4,5-trisphosphate receptor Ca2+-release channels), K+ channels (such as Ca2+-activated K+ channels, inward and outward K+ currents, and ATP-sensitive K+ channels), Na+ channels, and Cl- channels; 2) ion pumps, such as sarcoplasmic reticulum and sarcolemmal Ca2+ pumps, Na+-K+-ATPase (Na+ pump), and H+-ATPase (H+ pump); 3) ion exchangers such as the Na+/Ca2+ exchanger and Na+/H+ exchanger; and 4) ion cotransporters such as K+-Cl-, Na+-K+-Cl-, and Pi-Na+ cotransporters. The mechanism of ROS-induced modifications in ion transport pathways involves 1) oxidation of sulfhydryl groups located on the ion transport proteins, 2) peroxidation of membrane phospholipids, and 3) inhibition of membrane-bound regulatory enzymes and modification of the oxidative phosphorylation and ATP levels. Alterations in the ion transport mechanisms lead to changes in a second messenger system, primarily Ca2+ homeostasis, which further augment the abnormal electrical activity and distortion of signal transduction, causing cell dysfunction, which underlies pathological conditions.

Effects of ATP-sensitive potassium channel regulators on chloride channels in the sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl- channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 microM nor GABA at 150 microM had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with Ki approximately 30 microM. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, Imax and average mean current, I') and kinetic parameters (frequency of opening Fo, probability of the channel being open Po and mean open time, To, of the SCl channel. Glibenclamide increased mean closed time, Tc, and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I', Po, and To in the absence of ATP and Mg2+ but it had no effect on Imax and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I' followed by an inhibition of Imax, whereas cromakalim reduced Po and I' by increasing channel transitions to the closed state and reducing To without affecting Imax. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP]cis or [glibenclamide]cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a "flicker" type block mechanism. Modulation of the SCl channel by [diazoxide]cis and [glibenclamide]cis highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel.