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PubMed2004

A unified approach to burst properties of multiconductance single ion channels.

Patch clamp recordings from ion channels often show bursting behaviour, that is periods of repetitive activity, which are noticeably separated from each other by periods of inactivity. In this paper, bursting behaviour is considered for a general finite state space continuous-time Markov chain model of channel gating, allowing for one or more non-zero conductance levels. A unified semi-Markov framework, that encompasses both theoretical and empirical bursts, is described for analysing a broad range of properties of bursts, including the total charge transfer, the number of sojourns at distinct conductance levels and the number of openings in a burst, with the results presented when the channel is in equilibrium. When the gating mechanism is time reversible, it is shown that the distribution and autocorrelation function of each of the above properties are necessarily finite linear combinations of exponentially or geometrically decaying components with non-negative coefficients. Three methods for choosing a critical time for empirical bursts are investigated. The theory is illustrated by numerical examples from ryanodine, chloride and nicotinic acetylcholine receptor channels, demonstrating the power and flexibility of the methodology, and permitting comparison between the methods for choosing the critical time.

Electrophysiology↗
PubMed2003

C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening.

Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli. We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities. Application of Ni(2+) to the YggB-His(6) channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities. Western blot revealed that YggB-His(6) oligomers are more stable in the presence of Ni(2+), providing evidence that Ni(2+) is coordinated between C termini from different subunits of the channel. Intersubunit coordination of Ni(2+) affecting channel activities occurred in the channel closed conformation and not in the open state. This may suggest that the C termini move apart upon channel opening and are involved in the channel activation. We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel. The results are discussed and interpreted based on the recently released quaternary structure of the channel.

Amino Acid Sequence↗
PubMed1996

Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers.

Cholinergic synaptic vesicles were isolated from the electric organ of Torpedo californica. Vesicle membrane proteins were reconstituted into planar lipid bilayers by the nystatin/ergosterol fusion technique. After fusion, a variety of ion channels were observed. Here we identify four channels and describe two of them in detail. The two channels share a conductance of 13 pS. The first is anion selective and strongly voltage dependent, with a 50% open probability at membrane potentials of -15 mV. The second channel is slightly cation selective and voltage independent. It has a high open probability and a subconductance state. A third channel has a conductance of 4-7 pS, similar to the subconductance state of the second channel. This channel is fairly nonselective and has gating kinetics different from those of the cation channel. Finally, an approximately 10-pS, slightly cation selective channel was also observed. The data indicate that there are one or two copies of each of the above channels in every synaptic vesicle, for a total of six channels per vesicle. These observations confirm the existence of ion channels in synaptic vesicle membranes. It is hypothesized that these channels are involved in vesicle recycling and filling.

Animals↗
PubMed1994

A voltage-sensitive cation channel present in clusters in lobster skeletal muscle membrane.

The single channel properties of a voltage-sensitive cation channel are described in a study of ion channel activity in enzymatically induced blebs of lobster skeletal muscle membrane. This cation channel, one of several that are spontaneously active in excised patches from bleb membrane, can be distinguished from other channels on the basis of its large single channel conductance (293 pS), voltage-sensitive gating properties, the presence of a subconductance state of the fully open channel, and a weak selectivity for K > Na. At hyperpolarizing voltages, this channel displays flickering or bursting behavior, and a single state of the fully open channel is observed. At depolarizing voltages, the mean channel open time increases and a second longer-lived open state is observed. The voltage dependence of the mean channel open time and the linear i-V relation of this channel predict that the macroscopic current carried through this cation channel would be outwardly rectifying. Channels of this type are infrequently observed in this preparation, but when present in the patch are often present in multiple copies. We describe a statistical test for examining the clustering of ion channels in excised patches of membrane. The result of this test shows that the cation channels appear in clusters in the blebs.

Animals↗
PubMed1995

Evidence for a cGMP gated cation channel in photoreceptor cell membranes of Sepia officinalis.

It is assumed that cyclic nucleotides are involved in signal transduction of invertebrate photoreceptors. In this study, membranes of photoreceptor cells from freshly caught cuttlefish were isolated, and the membrane proteins were reconstituted into proteoliposomes. With the dye Neutral red it was possible to measure cyclic nucleotide induced Na+ fluxes into the liposomes. cGMP and cAMP concentrations for half maximal activation of Na+ fluxes are 77 microM and 224 microM, respectively, with Hill coefficients of 2.0 for cGMP and 2.4 for cAMP. These fluxes may demonstrate the presence of at least one cyclic nucleotide gated cation channel in the membranes of the photoreceptor cells of the invertebrate Sepia officinalis.

Animals↗
PubMed2004

Polycystins: from mechanosensation to gene regulation.

Polycystin proteins have been suggested to form mechanosensory transduction complexes involved in a variety of biological functions including sperm fertilization, mating behavior, and asymmetric gene expression in different species. Furthermore, their dysfunction is the cause of cyst formation in human kidney disease. This review focuses on the pros and cons of their candidacy as mechanically gated channels and on recent findings that have significantly advanced our physiological insight.

Animals↗
PubMed2000

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat.

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.

Adenosine Triphosphate↗
PubMed2003

Gating of MscL studied by steered molecular dynamics.

Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.

Binding Sites↗
PubMed2002

Open channel structure of MscL and the gating mechanism of mechanosensitive channels.

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

Computer Simulation↗
PubMed2001

Cyclic nucleotide-gated channels contribute to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons.

Plateau potentials are prolonged membrane depolarizations that are observed in hippocampal pyramidal neurons when spiking and Ca(2+) entry occur in combination with muscarinic receptor activation. In this study, we used whole-cell voltage clamping to study the current underlying the plateau potential and to determine the cellular signaling pathways contributing to this current. When combined with muscarinic stimulation, depolarizing command potentials that evoked Ca(2+) influx elicited a prolonged tail current (I(tail)) that had an extrapolated reversal potential of -20 mV. I(tail) was not observed when intracellular Ca(2+) levels were chelated with 10 mm intracellular BAPTA, and I(tail) was reversibly depressed in low external sodium. When I(tail) was evoked at intervals >3 min, current amplitudes were stable for up to 1 hr. However, at shorter intervals, I(tail) was refractory, with a time constant of recovery of 43.5 sec. The inhibitors of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and 6-anilino-5,8-quinolinequinone depressed I(tail) and zaprinast, which blocks cGMP-specific phosphodiesterase, enhanced I(tail), suggesting that a component of I(tail) was activated by cGMP. The inhibitors of cyclic nucleotide-gated (CNG) channels l-cis-diltiazem and 2',4'-dichlorobenzamil reversibly depressed I(tail). However, protein kinase G inhibition had no effect. Therefore, these results indicate that a component of I(tail) is attributable to activation of CNG channels. We conclude that Ca(2+) influx when combined with muscarinic receptor activation activates soluble guanylate cyclase and increases cGMP levels. The increased cGMP activates CNG channels and leads to prolonged depolarization. The cation conductance of the CNG channel contributes to the prolonged depolarization of the plateau potential.

Animals↗
PubMed1995

The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin.

Dynorphin is one of the endogenous opioids that modulates the excitability of nociceptive (pain-sensing) neurons. We have shown recently that dynorphin blocks NMDA-activated currents directly without the participation of kappa-opioid receptors. In order to understand the mechanism underlying this novel action of dynorphin, we examined, in detail, the interactions between dynorphin and NMDA receptors in isolated trigeminal neurons. Dynorphin reversibly blocks NMDA-activated current (INMDA). The onset and recovery of the block were determined with concentration jump experiments. The association rate (k+) of dynorphin(1-17) is 4.9 x 10(6) sec-1 M-1 and the dissociation rate (k-) is 7.5 sec-1. The apparent dissociation constant (KD) of dynorphin, calculated from these rate constants, is 1.6 microM. Dynorphin does not change the EC50 of NMDA, nor the potentiating action of glycine. The binding site for dynorphin is distinct from that of Zn2+ or H+. Upon treatment with the disulfide reducing agent dithiothreitol (DTT), NMDA receptors become less susceptible to dynorphin block. The affinity of dynorphin for the modified NMDA receptors is reduced by 2.7-fold. In analyses of single NMDA channels in cell-free patches, we found that dynorphin shortens the mean open time, decreases the probability of opening of NMDA channels, but has no effect on the single channel conductance. These results suggest that dynorphin interacts with a site conformationally linked with the redox site(s) on the NMDA receptor, thus altering the gating properties of the channel.

Animals↗
PubMed1997

Cyclic GMP-gated channels in a sympathetic neuron cell line.

The stimulation of IP3 production by muscarinic agonists causes both intracellular Ca2+ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3',5'-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide-gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na+ is 47 pS and is independent of voltage in the range -50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent KD of 10 microm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 microm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein-coupled neurotransmitter receptors can directly modify Ca2+ influx and electrical excitability. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refill the IP3-sensitive intracellular Ca2+ pool during repeated stimulation and CNG channels may play a similar role in other neurons.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons.

The importance of long-term synaptic plasticity as a cellular substrate for learning and memory is well established. By contrast, little is known about how learning and memory are regulated by voltage-gated ion channels that integrate synaptic information. We investigated this question using mice with general or forebrain-restricted knockout of the HCN1 gene, which we find encodes a major component of the hyperpolarization-activated inward current (Ih) and is an important determinant of dendritic integration in hippocampal CA1 pyramidal cells. Deletion of HCN1 from forebrain neurons enhances hippocampal-dependent learning and memory, augments the power of theta oscillations, and enhances long-term potentiation (LTP) at the direct perforant path input to the distal dendrites of CA1 pyramidal neurons, but has little effect on LTP at the more proximal Schaffer collateral inputs. We suggest that HCN1 channels constrain learning and memory by regulating dendritic integration of distal synaptic inputs to pyramidal cells.

Animals↗
PubMed1997

Regulation of metabolite flux through voltage-gating of VDAC channels.

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, "closed" state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5-11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically.

Ion Channel Gating↗
PubMed2000

Putting ion channels to work: mechanoelectrical transduction, adaptation, and amplification by hair cells.

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

Adaptation, Physiological↗
PubMed2001

Two-dimensional kinetic analysis suggests nonsequential gating of mechanosensitive channels in Xenopus oocytes.

Xenopus oocytes express mechanosensitive (MS(XO)) channels that can be studied in excised patches of membrane with the patch-clamp technique. This study examines the steady-state kinetic gating properties of MS(XO) channels using detailed single-channel analysis. The open and closed one-dimensional dwell-time distributions were described by the sums of 2-3 open and 5-7 closed exponential components, respectively, indicating that the channels enter at least 2-3 open and 5-7 closed kinetic states during gating. Dependency plots revealed that the durations of adjacent open and closed intervals were correlated, indicating two or more gateway states in the gating mechanism for MS channels. Maximum likelihood fitting of two-dimensional dwell-time distributions to both generic and specific models was used to examine gating mechanism and rank models. A kinetic scheme with five closed and five open states, in which each closed state could make a direct transition to an open state (two-tiered model) could account for the major features of the single-channel data. Two-tiered models that allowed direct transitions to subconductance open states in addition to the fully open state were also consistent with multiple gateway states. Thus, the gating mechanism of MS(XO) channels differs from the sequential (linear) gating mechanisms considered for MS channels in bacteria, chick skeletal muscle, and Necturus proximal tubule.

Animals↗
PubMed1994

Voltage-dependent gating of single gap junction channels in an insect cell line.

De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely.

Aedes↗
PubMed2000

[Activation of mechanosensitive ion channels in plasma membrane of K562 cells].

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.

Humans↗