Single-channel Gating Kinetics Research Articles

Shisa7-Dependent Regulation of GABAA Receptor Single-Channel Gating Kinetics.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABAAR-associated transmembrane proteins. We previously identified Shisa7 as a GABAAR auxiliary subunit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABAAR single-channel properties has yet to be determined. Here, we performed single-channel recordings of α2β3γ2L GABAARs cotransfected with Shisa7 in HEK293T cells and found that while Shisa7 does not change channel slope conductance, it reduced the frequency of receptor openings. Importantly, Shisa7 modulates GABAAR gating by decreasing the duration and open probability within bursts. Through kinetic analysis of individual dwell time components, activation modeling, and macroscopic simulations, we demonstrate that Shisa7 accelerates GABAAR deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABAAR gating and reveal for the first time that GABAAR single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission.SIGNIFICANCE STATEMENT Although GABAA receptor (GABAAR) single-channel properties are largely determined by pore-forming subunits, it remains unknown whether they are also controlled by GABAAR-associated transmembrane proteins. Here, we show that Shisa7, a recently identified GABAAR auxiliary subunit, modulates GABAAR activation by altering single-channel burst kinetics. These results reveal that Shisa7 primarily decreases the duration and open probability of receptor burst activity during gating, leading to accelerated GABAAR deactivation. These experiments are the first to assess the gating properties of GABAARs in the presence of an auxiliary subunit and provides a kinetic basis for how Shisa7 modifies temporal attributes of GABAergic transmission at the single-channel level.

Current-direction/amplitude-dependent single channel gating kinetics of mouse pannexin 1 channel: a new concept for gating kinetics

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl− influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl− efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl− with gluconate− diminishes the inward tail current (Cl− efflux) at a membrane potential of −100 mV due to the lowered outward current (gluconate− influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

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.

Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating.

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.

A mechanistic description of gating of the human cardiac ryanodine receptor in a regulated minimal environment

Cardiac muscle contraction, triggered by the action potential, is mediated by the release of Ca2+ from the sarcoplasmic reticulum through ryanodine receptor (RyR)2 channels. In situ, RyR2 gating is modulated by numerous physiological and pharmacological agents, and altered RyR2 function underlies the occurrence of arrhythmias in both inherited and acquired diseases. To understand fully the mechanisms underpinning the regulation of RyR2 in the normal heart and how these systems are altered in pathological conditions, we must first gain a detailed knowledge of the fundamental processes of RyR2 gating. In this investigation, we provide key novel mechanistic insights into the physical reality of RyR2 gating revealed by new experimental and analytical approaches. We have examined in detail the single-channel gating kinetics of the purified human RyR2 when activated by cytosolic Ca2+ in a stringently regulated environment where the modulatory influence of factors external to the channel were minimized. The resulting gating schemes are based on an accurate description of single-channel kinetics using hidden Markov model analysis and reveal several novel aspects of RyR2 gating behavior: (a) constitutive gating is observed as unliganded opening events; (b) binding of Ca2+ to the channel stabilizes it in different open states; (c) RyR2 exists in two preopening closed conformations in equilibrium, one of which binds Ca2+ more readily than the other; (d) the gating of RyR2 when bound to Ca2+ can be described by a kinetic scheme incorporating bursts; and (e) analysis of flicker closing events within bursts reveals gating activity that is not influenced by ligand binding. The gating schemes generated in this investigation provide a framework for future studies in which the mechanisms of action of key physiological regulatory factors, disease-linked mutations, and potential therapeutic compounds can be described precisely.

How do Mutations in M0 of KCNJ11 Produce Diabetes?

Many diabetogenic mutations (Endocrine Rev 2009;29:265) map to the canonical, N-terminal, submembrane (BBRC 1999;255:231) “slide” M0 helix of KCNJ11 (KIR6.2). To clarify the principal biophysical mechanism of their action, inhibiting insulin release, I analyzed effects of the first reported severe Neonatal Diabetes (ND with epilepsy and developmental delay) mutation and 18 other mutations, V59X, in M0 on macroscopic and unitary currents through ABCC8(SUR1)-containing ATP-sensitive potassium (KATP) channels, reconstituted in mammalian cells lacking endogenous SUR or KIR. Several V59X decreased, and no V59X increased or abolished, functional expression (N) of the neuroendocrine-type adenine nucleotide sensors. This a) indicated that the effect of any possible ND V59X-induced decrease in N on Vm in humans is overruled by the mean open channel probability(PO)-increasing effect, explaining insufficient insulin release due to hyperpolarization of insulin producing cells, and b) allowed complete analysis of relationships between the physical properties of the side chain in the middle of M0 and PO, its sensitivity to nucleotides, and single-channel gating kinetics. The established relationships are consistent with the results of molecular modeling and molecular dynamics simulation of severe ND KATP pores and strongly suggest that a ligand-independent stabilization of the active (burst) state with conformations without specific, micromolar affinity for inhibitory ATP, is the principal mechanism of pathogenic hyperactivity of KATP with mutations in M0, the small domain proposed to play a big role in gating of KIR6 and their relatives.I thank the National Institutes of Health for funding and G. Zhao for technical assistance.

Ca2+ Activates TRPM2 Channels By Binding In Deep Crevices Near The Pore, But Intracellularly Of The Gate

TRPM2 is a tetrameric Ca2+ permeable channel involved in immunocyte respiratory burst and postischaemic neuronal death. In whole cells TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding site(s) for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages, and Ca2+ dependence of steady-state single-channel gating kinetics, provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus oocytes expressing human TRPM2, co-application of intracellular ADPR and Ca2+ activated ∼50-pS non-selective cation channels; K1/2 for ADPR was ∼1μM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open- than in the closed-channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1mM free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells which contain micromolar ADPR even brief Ca2+ spikes likely trigger prolonged, self-sustained TRPM2 activity.

Interaction Between Two Domains in the AChR Gating Reaction

The AChR is a large ion channel that isomerizes between non-conducting and conducting conformations. Residue αA96 is located in loop 5 (loop A) near the agonist-binding site, which moves at the outset of the channel-opening process (the Φ value for the adjacent residue αD97 is 0.93). Side chain substitutions at nearby (<0.45 nm, 2QC1.pdb) residue αY127 (β-strand 6) change the gating equilibrium constant (Keq) by up to 290,000-fold (Φ=0.77). This suggests that αY127 moves in concert with the lower part of the extracellular domain, after the motion of loop 5. αD97 and αY127 are not coupled energetically. We tested the hypothesis that αA96 and αY127 energetically link the first two Φ-blocks, to propagate the opening conformational wave from the upper to the lower part of the extracellular domain. We mutated residue αA96 (C, F, K, L, N, Q) and measured single-channel gating kinetics (mouse α2βδe, cell-attached, -100 mV, 20 mM choline, PBS, 23°C). The Φ-value for αA96 is 0.90, indicating that it moves at the onset of channel gating along with other residues in loop 5. αA96N showed the largest change in Keq (∼900-fold) and markedly increased unliganded gating. Next, we performed mutant cycle analysis to test for energetic coupling between αA96 and αY127. Keq for the double mutant αA96K+αY127E is 18-fold greater than the wt, where the effects of the single mutants, if additive, predict one that is 9.2-fold smaller. This corresponds to a coupling free energy of -3.1 kcal/mol. Similarly, Keq for the double mutant αA96C+αY127C is 213-fold greater than the wt, whereas a value 1.7-fold smaller is predicted assuming independence (coupling free energy of -3.6 kcal/mol). These are large interaction energies that suggest αA96 and αY127 form a key energetic link between the first and second Φ-blocks.

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

TRPM2 is a tetrameric Ca2+-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding sites for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca2+ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ∼50-pS nonselective cation channels; K1/2 for ADPR was ∼1 µM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1 mM of free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca2+ likely triggers prolonged, self-sustained TRPM2 activity.

Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

The α1 and α6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kinetics

The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The α family is the largest, with six different subtypes. The α1 and α6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the α1 or α6 subunit was expressed in mammalian cells along with β3 and γ2L subunits and the kinetic properties examined with outside‐out patch recordings. The α1β3γ2L receptors responded to GABA with long‐duration openings organized into multi‐opening bursts. In contrast, channel openings of the α6β3γ2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2‐fold slower for α6β3γ2L, compared with the α1β3γ2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the α1 and α6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3–TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N‐terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore‐lining domains are responsible for the kinetic differences conferred by the α1 and α6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition.

ATP-dependent adenophostin activation of inositol 1,4,5-trisphosphate receptor channel gating: kinetic implications for the durations of calcium puffs in cells.

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.

Structure-function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes.

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Deltagm, was initially determined. DT-AB induced a approximately 10-fold lower Deltagm than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Deltagm, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Deltagm. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Deltagm and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.

Evaluation of single-channel gating kinetics produced after amplitude-based separation of unitary currents

Cell-attached patch-clamp recording has been established as a major technique for investigating ion channel behaviour in a physiological setting, despite the problems which arise in analysing records containing more than one type of unitary current. To circumvent these problems, single-channel amplitude-based assignment of discrete single-channel events to different channel types becomes increasingly necessary. Surprisingly, a systematic evaluation of the validity of this method in determining single-channel parameters has not been performed to date. Using computer-simulated single-channel traces, and recordings from a biological preparation containing well-characterized ion channels (N1E-115 neuroblastoma cells), we have explored the accuracy by which amplitude-based separation recovers ion-channel parameters. Determination of gating kinetics after separation revealed that even a very small contamination in the selected population yields additional time constants in the probability density functions. Therefore, our results demonstrate that, whereas the use of amplitude-based separation is straightforward for determining slope conductance and reversal potential, it is prone to incorporate errors in establishing gating kinetics. Ways of identifying such errors are described.

S4 mutations alter the single-channel gating kinetics of Shaker K + channels

The S4 sequence comprises at least part of the voltage sensor in Shaker K + channels. Two site-directed mutations in the Shaker S4 sequence, R368Q and R377K, which decrease the voltage dependence of the whole-cell current, alter voltage-dependent gating at the single-channel level. Compared with the wild-type channel, they increase the latency to first opening, destabilize the open state, and alter the equilibria of voltage-dependent transitions, so that some of the charge movement occurs after the first opening. Whether these changes reflect a new mechanism of activation is a key question. Although the mutations alter the kinetics of many steps in the gating process, we conclude that the mutant channels are likely to activate using the same fundamental mechanism as wild-type channels.