Open Channel Lifetime Research Articles

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites.Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

α4β2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL

Acetylcholine receptors comprising α4 and β2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and β2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or β2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4β2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4β2)2β2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and β2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties.

Tyrosine Residue in the TRPV1 Vanilloid Binding Pocket Regulates Deactivation Kinetics

Vanilloids are pain evoking molecules that serve as ligands of the "heat and capsaicin receptor" TRPV1. Binding of either endogenous or exogenous vanilloids evokes channel and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation and deactivation is not fully understood. The highly conserved tyrosine in position 511 (Tyr(511)) of the rat TRPV1 (rTRPV1) was the first residue to be identified as a necessary participant in the vanilloid-mediated response. rTRPV1 cryo-EM structures implicated rotation of this residue in the vanilloids bound state. Therefore, we hypothesize that the rTRPV1 Tyr(511) residue entraps vanilloids in their binding site, prolonging channel activity. To test our hypothesis, we generated an array of rTRPV1 mutants, containing the whole spectrum of Tyr(511) substitutions, and tested their response to both exo- and endovanilloids. Our data show that only substitutions of Tyr(511) to aromatic amino acids were able to mimic, albeit partially, the vanilloid-evoked activation pattern of the wt receptor. Although these substitutions reduced the channel sensitivity to vanilloids, a maximal open-channel lifetime could be achieved. Moreover, whereas their current activation rate remains intact, receptors with Tyr(511) substitutions exhibited a faster current deactivation. Our findings therefore suggest that the duration of channel activity evoked by vanilloids is regulated by the interaction between Tyr(511) and the agonist. To conclude, we suggest that Tyr(511)-mediated anchoring of vanilloids in their binding pocket is pivotal for TRPV1 activation and subsequent pain sensation.

Exploring the positive allosteric modulation of human α7 nicotinic receptors from a single-channel perspective

Enhancement of α7 nicotinic receptor (nAChR) function by positive allosteric modulators (PAMs) is a promising therapeutic strategy to improve cognitive deficits. PAMs have been classified only on the basis of their macroscopic effects as type I, which only enhance agonist-induced currents, and type II, which also decrease desensitization and reactivate desensitized nAChRs. To decipher the molecular basis underlying these distinct activities, we explored the effects on single-α7 channel currents of representative members of each type and of less characterized compounds. Our results reveal that all PAMs enhance open-channel lifetime and produce episodes of successive openings, thus indicating that both types affect α7 kinetics. Different PAM types show different sensitivity to temperature, suggesting different mechanisms of potentiation. By using a mutant α7 receptor that is insensitive to the prototype type II PAM (PNU-120596), we show that some though not all type I PAMs share the structural determinants of potentiation. Overall, our study provides novel information on α7 potentiation, which is key to the ongoing development of therapeutic compounds.

The Stoichiometry and Activation Mechanism of TRPV1 by Vanilloids

Vanilloids are pain evoking molecules that serve as ligands of the ‘heat and capsaicin receptor’ TRPV1. It is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat (>42°C) and various noxious chemicals. TRPV1 channel is formed by the symmetrical arrangement of four identical subunits around a central ion-conducting pore. Binding of vanilloids evokes channel opening and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation by vanilloids is not fully understood. Here, we combine the use of site-directed mutagenesis, concatemeric constructs harboring mutated binding sites with patch-clamp recordings and calcium imaging in order to determine the stoichiometry and activation mechanism of TRPV1 by the exo-vanilloid capsaicin, the ‘hot’ component of chili peppers. We show that a single capsaicin-bound subunit is sufficient to achieve a maximal open-channel lifetime, while the high sensitivity for this agonist requires all four subunits. Moreover, we defined the role of residues within TRPV1 vanilloid binding site in the channel activation mechanism, by acting to stabilize ligand-receptor interaction.

The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.

The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains.

The coupling interface and pore domain codetermine the single-channel activity of the α7 nicotinic receptor

Ligand-gated ion channels play a role in mediating fast synaptic transmission for communication between neurons. However, the structural basis for the functional coupling of the binding and pore domains, resulting in channel opening, remains a topic of intense investigation. Here, a series of α7 nicotinic receptor mutants were constructed for expression in cultured mammalian cells, and their single-channel properties were examined using the patch-clamp technique combined with radio ligand binding and the fluorescence staining technique. We demonstrated that the replacement of the four pore-lining residues in the channel domain of the α7 nicotinic receptor with the hydrophilic residue serine prolongs the open-channel lifetime, although the conductance of these mutants decreases. At the coupling interface between the extracellular and transmembrane domains, when the VRW residues in the Cys-loop were substituted with the corresponding residues (i.e., IYN) in the 5-HT3A receptor, the single-channel activity elicited by acetylcholine is impaired. This effect occurred despite the expression of the mutant receptors on the cell surface and despite the fact that the apparent Kd values were much lower than those of the wild-type α7 receptor. When we further lowered the channel-gating barrier of this chimera to enhance the open-channel probability, the loss of function was rescued. Overall, we explored the microscopic mechanisms underlying the interplay between the channel domains and the coupling interface that affect the channel activity, and we generated an allosteric gating model for the α7 receptor. This model shows that the gating machinery and coupling assembly codetermine the single-channel gating kinetics. These results likely apply to all channels in the Cys-loop receptor family.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

Stoichiometry for activation of neuronal α7 nicotinic receptors

Neuronal α7 nicotinic receptors elicit rapid cation influx in response to acetylcholine (ACh) or its hydrolysis product choline. They contribute to cognition, synaptic plasticity, and neuroprotection and have been implicated in neurodegenerative and neuropsychiatric disorders. α7, however, often localizes distal to sites of nerve-released ACh and binds ACh with low affinity, and thus elicits its biological response with low agonist occupancy. To assess the function of α7 when ACh occupies fewer than five of its identical binding sites, we measured the open-channel lifetime of individual receptors in which four of the five ACh binding sites were disabled. To improve the time resolution of the inherently brief α7 channel openings, background mutations or a potentiator was used to increase open duration. We find that, in receptors with only one intact binding site, the open-channel lifetime is indistinguishable from receptors with five intact binding sites, counter to expectations from prototypical neurotransmitter-gated ion channels where the open-channel lifetime increases with the number of binding sites occupied by agonist. Replacing the membrane-embedded domain of α7 by that of the related 5-HT3A receptor increases the number of sites that need to be occupied to achieve the maximal open-channel lifetime, thus revealing a unique interdependence between the detector and actuator domains of these receptors. The distinctive ability of a single occupancy to elicit a full biological response adapts α7 to volume transmission, a prevalent mechanism of ACh-mediated signaling in the nervous system and nonneuronal cells.

Contribution of Agonist Binding Sites and Coupling Regions to Activation and Desensitization of Homomeric Cys-Loop Receptors

Homomeric receptors are the simplest structural class of receptors of the Cys-loop superfamily and are therefore invaluable models for probing fundamental relationships between structure and function. To determine how many of the five agonist binding sites are required to be occupied by the agonist for maximal open channel stability we applied an electrical fingerprinting strategy in the homomeric receptor model, α7-5HT3A. To vary the number of functional agonist binding sites we installed mutations that prevent agonist binding, and to report the presence of the mutant subunit, we installed mutations that alter single-channel conductance. We find that receptors can be activated by occupancy of only one agonist binding site but open channel lifetime is brief, and occupancy of three non-consecutive sites is required for maximal open channel lifetime. The conformational changes initiated at the binding site are propagated to the gate through the extracellular-transmembrane interface, known as coupling region. We show that structural differences in the coupling region of homomeric α7 and 5-HT3A receptors account for the large differences in open channel lifetime and rate of desensitization between these homomeric members of the superfamily. By applying the electrical fingerprinting strategy, we determine that each coupling region in the pentamer contributes an equal increment to the stability of the open channel. We also determine minimal requirements for channel opening regardless of stability of the open state, and find that receptors can open with one functional binding site and two contiguous and functional coupling regions, or with five functional binding sites and only one functional coupling region. The overall findings show that whereas the agonist binding sites contribute inter-dependently and asymmetrically to open channel stability, the coupling regions contribute independently and symmetrically.

New insights into the structural bases of activation of Cys-loop receptors

Neurotransmitter receptors of the Cys-loop superfamily mediate rapid synaptic transmission throughout the nervous system, and include receptors activated by ACh, GABA, glycine and serotonin. They are involved in physiological processes, including learning and memory, and in neurological disorders, and they are targets for clinically relevant drugs. Cys-loop receptors assemble either from five copies of one type of subunit, giving rise to homomeric receptors, or from several types of subunits, giving rise to heteromeric receptors. Homomeric receptors are invaluable models for probing fundamental relationships between structure and function. Receptors contain a large extracellular domain that carries the binding sites and a transmembrane region that forms the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50Å to the ion channel gate is central to understanding receptor function. Depending on the receptor subtype, occupancy of either two, as in the prototype muscle nicotinic receptor, or three binding sites, as in homomeric receptors, is required for full activation. The conformational changes initiated at the binding sites are propagated to the gate through the interface between the extracellular and transmembrane domains. This region forms a network that relays structural changes from the binding site towards the pore, and also contributes to open channel lifetime and rate of desensitization. Thus, this coupling region controls the beginning and duration of a synaptic response. Here we review recent advances in the molecular mechanism by which Cys-loop receptors are activated with particular emphasis on homomeric receptors.

Self-assembled, cation-selective ion channels from an oligo(ethylene glycol) derivative of benzothiazole aniline

This paper describes the spontaneous formation of well-defined pores in planar lipid bilayers from the self-assembly of a small synthetic molecule that contains a benzothiazole aniline (BTA) group attached to a tetra-ethylene glycol (EG4) moiety. Macroscopic and single-channel current recordings suggest that these pores are formed by the assembly of four BTA-EG4 monomers with an open pore diameter that appears similar to the one of gramicidin pores (~0.4nm). The single-channel conductance of these pores is modulated by the pH of the electrolyte and has a minimum at pH ~3. Self-assembled pores from BTA-EG4 are selective for monovalent cations and have long open channel lifetimes on the order of seconds. BTA-EG4 monomers in these pores appear to be arranged symmetrically across both leaflets of the bilayer, and spectroscopy studies suggest that the fluorescent BTA group is localized inside the lipid bilayers. In terms of biological activity, BTA-EG4 molecules inhibited growth of gram-positive Bacillus subtilis bacteria (IC50 ~50μM) and human neuroblastoma SH-SY5Y cells (IC50 ~60μM), while they were not toxic to gram-negative Escherichia coli bacteria at a concentration up to 500μM. Based on these properties, this drug-like, synthetic, pore-forming molecule with a molecular weight below 500gmol−1 might be appealing as a starting material for development of antibiotics or membrane-permeating moieties for drug delivery. From a biophysical point of view, long-lived, well-defined ion-selective pores from BTA-EG4 molecules offer an example of a self-assembled synthetic supramolecule with biological function.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

Electrical Fingerprinting Reveals Agonist Binding Sites Required for Activation of Homo-pentameric Cys-loop Receptors

Ancestral neurotransmitter Cys-loop receptors were homo-pentamers harboring five identical agonist binding sites but most present day receptors are hetero-pentamers with only two binding sites. To understand why Cys-loop receptors evolved to utilize fewer than five binding sites, we disabled different numbers of sites and developed a method to monitor lifetimes of individual active receptors and the corresponding number of functional binding sites. We find that maximal open-channel lifetime is achieved when the neurotransmitter occupies three non-consecutive binding sites. Occupancy of one site allows receptor activation, although the open state is unstable; occupancy of two non-consecutive sites produces a much longer-lived open state appropriate for efficient activation. However, occupancy of a third site further increases channel lifetime, thus providing optimal stabilization of the active state. Maximal activation of homomeric receptors by agonist occupancy of less than the five potential sites enhances the rate of channel opening and increases agonist sensitivity. The results reveal that allosteric requirements dictated the number and location of the agonist binding sites, and provide an indispensable framework for further progress in drug design.

The Interface between Extracellular and Transmembrane Domains of Homomeric Cys-Loop Receptors Governs Open-Channel Lifetime and Rate of Desensitization

The lifetimes of activated postsynaptic receptor channels contribute to the efficiency of synaptic transmission. Here we show that structural differences within the interface dividing extracellular and transmembrane domains of homomeric alpha7 and 5-HT(3A) receptors account for the large differences in open-channel lifetime and time of desensitization onset between these contrasting members of the Cys-loop receptor superfamily. For alpha7 receptors, agonist-evoked single-channel currents appear mainly as isolated brief openings (tau(o) = 0.35 ms), whereas macroscopic currents after a step pulse of agonist desensitize rapidly (tau(d) = 0.4 ms). In contrast for 5-HT(3A) receptors, agonist-evoked single-channel currents appear as clusters of many long openings in quick succession (tau(cluster) = 1.2 s), whereas macroscopic currents desensitize slowly (tau(d) = 1.1 s). A chimeric alpha7-5HT(3A) receptor exhibits functional properties intermediate between those of the parent receptors, but the functional signatures of each parent are reconstituted after substituting the major loops within the interface of the extracellular and transmembrane domains from the corresponding parent receptor. Furthermore, these structural loops contribute to open-channel lifetime and time of desensitization onset in a nonadditive manner. The results suggest that desensitization is the major determinant of the lifetimes of activated alpha7 and 5-HT(3A) receptors and that functional differences between the two receptors arise primarily through structural differences at the interface between extracellular and transmembrane domains.

Identification of gating modes in single native Na+ channels from human atrium and ventricle.

The aim of the present study was to investigate the single-channel properties of different gating modes in the native human cardiac Na+ channel. Patch-clamp experiments were performed at low noise using ultrathick-walled pipettes. In 17 cell-attached patches containing only one channel, fast back and forth switching between five different Na+-channel gating modes (F-mode, M1-mode, M2-mode, S-mode, and P-mode) was identified, but no difference in the gating properties was found between normal and diseased cardiomyocytes from atrium or ventricle, respectively. Hodgkin-Huxley fits to the ensemble-averaged currents yielded the activation-time (tau(m)) and inactivation-time (tau(h)) constants. tau(m) was comparably fast in the F-mode, M1-mode, M2-mode, and S-mode (0.15 ms) and slow in the P-mode (0.3 ms). tau(h) ranged from 0.35 ms (F-mode) to 4.5 ms (S-mode and P-mode). The mean open-channel lifetime (tau(o)) was shortest in the F-mode and P-mode (0.15 ms) and longest in the S-mode (1.25 ms). The time before which half of the first channel openings occurred (t(0.5)) was comparably short in the F-mode, M1-mode, M2-mode, and S-mode (0.3 ms) and long in the P-mode (0.9 ms). It is concluded that (1) a single native human cardiac Na+ channel can be recorded at low noise, (2) this channel can change its gating properties at a time scale of milliseconds, (3) lifetimes of the observed gating modes are short ranging from milliseconds to seconds only, and (4) the gating modes are characterized by specific activation and inactivation kinetics and differ at least in their mean open time and first latency.

The extracellular linker of muscle acetylcholine receptor channels is a gating control element.

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.

Activation and block of recombinant GABA(A) receptors by pentobarbitone: a single-channel study.

Recombinant GABA(A) receptors (alpha1beta2gamma2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell-attached single-channel patch clamp. Clusters of single-channel activity elicited by 500 microM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel opening rate constant is approximately 1500 s(-1), and the estimated equilibrium dissociation constants for the binding steps involved in activation are approximately 2 mM. Our results show a dose-dependent block of receptors at millimolar concentrations of PB that results in reduced open interval durations. The reduction in mean open time is linearly proportional to PB concentration, indicating that block can be produced by binding of a single PB molecule. Addition of millimolar concentrations of PB in the presence of GABA also produces a reduction of open channel lifetime in addition to a progressive increase in the closed interval durations within a cluster. The data suggest that the receptor contains two or more blocking sites while occupancy of only one of the sites is sufficient for channel block. Neither the blocking rate constant nor return rate from the blocked state(s) is affected by pH (ionization status of the PB molecule) demonstrating that both neutral and anionic forms of PB cause channel block.

Characterization of the driving force as a modulator of gating in cardiac ATP-sensitive K+ channels - evidence for specific elementary properties.

Single cardiac ATP-sensitive K+ channels and, comparatively, two other members of the inwardly rectifying K+ channel family, cardiac K+(ir) and K+(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K+ gradient for open-state kinetics of these K+ channels. The conductive state of K+(ATP) channels was defined as a function of the electrochemical K+ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) tauopen proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K+(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of tauopen. Since gating continued to operate at EK, i.e., in the absence of K+ permeation through the pore, K+ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb+ whose interference with an externally located binding site stabilizes the open state. In K+(ir) and K+(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K+ gradient. Thus, K+(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus.

A Distinct Contribution of the δ Subunit to Acetylcholine Receptor Channel Activation Revealed by Mutations of the M2 Segment

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12′ position of the M2 segment) were examined at the single-channel level. For all subunits ( α, β, ϵ, and δ), a 12′P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12′P mutations were co-expressed with α subunits having a binding site mutation that slows channel opening ( αD200N). In these AChRs, a 12′P mutation in ϵ or β slowed the closing rate constant ∼6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh ( K d). In contrast, a 12′P mutation in δ slowed the channel closing rate constant only ∼2-fold and significantly increased both the channel opening rate constant and the K d. Pairwise expression of 12′P subunits indicates that mutations in ϵ and β act nearly independently, but one in δ reduces the effect of a homologous mutation in ϵ or β. The results suggest that a 12′P mutation in ϵ and β has mainly local effects, whereas one in δ has both local and distributed effects that influence both agonist binding and channel gating.