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

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.     

Prion channel proteins and their role in vacuolation and neurodegenerative diseases

The prion encephalopathies, which are characterized by neuropathological changes that include vacuolation, astrocytosis, the development of amyloid plaques and neuronal loss, are associated with the conversion of a normal cellular isoform of prion protein (PrP(c)) to an abnormal pathologic scrapie isoform (PrP(Sc)). The use of PrP[106-126] and its isoforms in studies of channels in lipid bilayers has revealed that it forms heterogeneous channels reflecting modifications in the peptide's structure and differences in the properties of the formed oligomeric aggregates and their intermediates. We propose that the accumulation of pathological isoforms of prion are linked to membrane abnormalities and vacuolation in prion diseases. The interlinked changes in membrane fluidity and endogenous channels induced by prion isoforms can occur independently and concurrently with channel formation, i.e. they are not mutually exclusive. We suggest that vacuolation is a cellular response triggered in order to immobilize pathological prion isoforms having the ability to form channels that compromise cellular membranes. This mechanism is similar to that of other channel-forming proteins that induce vacuolation, e.g. the well-established VacA of Helicobacter pylori, Vero cells and aerolysin, as well as melittin-induced micellization and membrane fusion. We conclude that channel formation is part of the molecular mechanisms responsible for the vacuolation associated with prion diseases. The initial vacuolation could be an adaptive cellular response to compartmentalize the increase in pathogenic prion isoforms, while an excessive accumulation of pathologic prion isoforms in later stages represents the inability of the cell to continue to compartmentalize these misfolded proteins in vacuoles.     

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 (AP) 1–40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AP[1–40]-formed ion channels have been identified: (i) The AP[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 P _K > P _Na > P _Li = 1.0:0.60:0.47, (c) P_o 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 AP[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 P _Rb > P _K > P _Cs > P _Na > P _Li = 1.3:1.0:0.46:0.40:0.27. (iii) The AP[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 V_ms more negative than –120 and more positive than +120 mV. (iv) The AP[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 KCl) 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 P _K/P _choline for the fast mode was 3.9 and selectivity sequence P _K > P _Cs > P _Na > P _Li = 1.0:0.94:0.87:0.59. The value of P _K/P _choline for the slow mode was 2.7 and selectivity sequence P _K > P _Na > P _Li > P _Cs = 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 -sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.     

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 μm nor GABA at 150 μm 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 K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max 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 I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. 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. Received: 2 September 1997/Revised: 20 March 1998     

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 (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 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 pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis 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 pH _cis -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 pH _cis 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. Received: 20 May 1998/Revised: 24 September 1998     

Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A comparison is made of two types of chloride-selective channel in skeletal muscle sarcoplasmic reticulum (SR) vesicles incorporated into lipid bilayers. The I/V relationships of both channels, in 250/50 mM Cl- (cis/trans), were linear between -20 and +60 mV (cis potential,) reversed near Ecl and had slope conductances of approximately 250 pS for the big chloride (BCl) channel and approximately 70 pS for the novel, small chloride (SCl) channel. The protein composition of vesicles indicated that both channels originated from longitudinal SR and terminal cisternae. BCl and SCl channels responded differently to cis SO4(2-) (30-70 mM), 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (8-80 microM) and to bilayer potential. The BCl channel open probability was high at all potentials, whereas SCl channels exhibited time-dependent activation and inactivation at negative potentials and deactivation at positive potentials. The duration and frequency of SCl channel openings were minimal at positive potentials and maximal at -40 mV, and were stationary during periods of activity. A substate analysis was performed using the Hidden Markov Model (S. H. Chung, J. B. Moore, L. Xia, L. S. Premkumar, and P. W. Gage, 1990, Phil. Trans. R. Soc. Lond. B., 329:265-285) and the algorithm EVPROC (evaluated here). SCl channels exhibited transitions between 5 and 7 conductance levels. BCl channels had 7-13 predominant levels plus many more short-lived substates. SCl channels have not been described in previous reports of Cl- channels in skeletal muscle SR.     

Properties of cytotoxic peptide-formed ion channels

Cytotoxic peptides are relatively small cationicmolecules such as those found 1) in venoms, e.g., melittin inbee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, andlycotoxin 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) asautocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic-cells, prion peptide fragment 106-126[PrP-(106-126)], and amyloid -protein (AP)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 -sheets, e.g., mammalian defensins, AP, amylin, and PrP-(106-126),whereas the larger cytotoxic peptides have several domains composed of both -helices and -sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modifycell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These twononexclusive mechanisms of action lead to changes in second messengersystems that further augment the abnormal electrical activity anddistortion of the signal transduction causing cell death.

     

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 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.