Single-channel Kinetics Research Articles

ML277 regulates KCNQ1 single-channel amplitudes and kinetics, modified by voltage sensor state.

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell-derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

Sleep-related hypermotor epilepsy associated mutations uncover important kinetic roles of α4β2- nicotinic acetylcholine receptor intracellular structures.

Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains α4 and β2 subunits, in two ratios. (α4β2)2β2-nAChR have high agonist sensitivity (HS-isoform), whereas (α4β2)2α4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of α4 and β2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [α4(R336H) and β2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform α4β2-nAChR were expressed by injecting either 1:10 or 30:1 α4:β2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant α4β2-nAChR isoforms were compared to their wildtype counterparts. α4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas β2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The α4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the β2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (α3β4-nAChR and 5-HT3AR).

The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating

The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.

Subunit composition critically determines pH sensitivity of epithelial sodium channels in Xenopus laevis

The Epithelial Sodium Channel (ENaC) mediates the selective flux of sodium ions across the apical membrane of epithelial cells. ENaC consists of three subunits (α,β,γ) and its activity at the cell surface is subject to a multitude of regulatory mechanisms, including proteolytic cleavage, extracellular pH and Na+ ions. Channel regulation may also depend on ENaC subunit composition, as an additional δ‐subunit can replace α‐ENaC in the heterotrimeric configuration. As the physiological implications of ENaC containing the δ‐subunit are poorly understood, we characterized the regulation of Xenopus δβγ‐ENaC by extracellular pH and Na+.Regulation of Xenopus δβγ‐ENaC by extracellular Na+ and pH was investigated by two‐electrode voltage‐clamp and cell‐attached Patch‐Clamp electrophysiology using the Xenopus oocyte expression system. Successive reduction of extracellular pH from pH 8 – 6 increased amiloride‐sensitive transmembrane currents of oocytes expressing δβγ‐ENaC by > 7 fold. In cell‐attached Patch‐Clamp recordings the pH of the pipette solution (pHpip = extracellular) affected δβγ‐ENaC open probability and kinetics. Compared to pHpip at 7.4, alkaline conditions (pHpip 8) decreased open probability as well as the mean channel open time, whereas under acidic conditions (pHpip 6) an increased open probability was accompanied by a reduction of average channel closed times. Transmembrane currents mediated by Xenopus δβγ‐ENaC are sensitive to sodium self‐inhibition, a phenomenon describing the fast inhibition of ENaC in the presence of high extracellular Na+ concentrations. Channel activation under acidic conditions (pH 7 & 6) was accompanied by a decrease in magnitude and apparent Na+‐affinity of self‐inhibition, whereas these effects were reversed under alkaline conditions (pH 8). Hypothesizing mutual regulation of δβγ‐ENaC activity by extracellular Na+ and pH, we examined whether neutralization of pH‐sensitive aspartates in the δ‐subunit affected channel self‐inhibition and regulation by pH. Specific aspartates in a subunit domain corresponding to a putative Na+‐sensing site of mouse α‐ENaC (Kashlan et al., 2015, J Biol Chem 290:568–76) were replaced by asparagines via site‐directed mutagenesis (δD293N, δD296N, δD293,6N). All mutations decreased pH‐mediated channel activation. Notably, ENaC containing the δD296N mutation also displayed a decreased sodium self‐inhibition, which was not observed in channels bearing a single δD293N mutation. Furthermore, human δβγ‐ENaCs which have a reduced sodium self‐inhibition were not activated by acidic pH as observed with the Xenopus orthologue displaying a strong sodium self‐inhibition.In conclusion, these results demonstrate that activity of ENaC containing the δ‐subunit in Xenopus laevis is sensitive to changes in the extracellular pH, whereas ENaC containing the α‐subunit is not. Proton‐mediated activation of ENaC involves changes in single channel kinetics and a reduction of sodium self‐inhibition. Thus, sensitivity of this amphibian ENaC to extracellular pH is critically determined by its subunit composition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A common mechanism for CFTR potentiators.

Cystic fibrosis (CF) is a channelopathy caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a phosphorylation-activated and adenosine triphosphate (ATP)-gated chloride channel. In the past few years, high-throughput drug screening has successfully realized the first US Food and Drug Administration-approved therapy for CF, called ivacaftor (or VX-770). A more recent CFTR potentiator, GLPG1837 (N-(3-carbamoyl-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-3-carboxamide), has been shown to exhibit a higher efficacy than ivacaftor for the G551D mutation, yet the underlying mechanism of GLPG1837 remains unclear. Here we find that despite their differences in potency and efficacy, GLPG1837 and VX-770 potentiate CFTR gating in a remarkably similar manner. Specifically, they share similar effects on single-channel kinetics of wild-type CFTR. Their actions are independent of nucleotide-binding domain (NBD) dimerization and ATP hydrolysis, critical steps controlling CFTR's gate opening and closing, respectively. By applying the two reagents together, we provide evidence that GLPG1837 and VX-770 likely compete for the same site, whereas GLPG1837 and the high-affinity ATP analogue 2'-deoxy-N6-(2-phenylethyl)-adenosine-5'-O-triphosphate (dPATP) work synergistically through two different sites. We also find that the apparent affinity for GLPG1837 is dependent on the open probability of the channel, suggesting a state-dependent binding of the drug to CFTR (higher binding affinity for the open state than the closed state), which is consistent with the classic mechanism for allosteric modulation. We propose a simple four-state kinetic model featuring an energetic coupling between CFTR gating and potentiator binding to explain our experimental results.

Beyond cystic fibrosis transmembrane conductance regulator (CFTR) single channel kinetics: implications for therapeutic intervention.

Beyond cystic fibrosis transmembrane conductance regulator (CFTR) single channel kinetics: implications for therapeutic intervention.

In praise of single channel kinetics.

It is more than 30 years since the advent of the gigaohm seal patch clamp technique, which enabled routine recordings of currents generated by the activity of a single ion channel molecule in its native environment. The muscle nicotinic acetylcholine (ACh) receptor (nAChR) is one of the best ligand-

Cardiac Ryanodine Receptor Channel Gating is Modified during Blocker-Induced Changes in Ion Permeation

Ion channel blockers such as large quaternary ammonium (QA) compounds have traditionally been used as effective tools for looking at the interplay between ion permeation, channel gating and block in various voltage/ligand-gated K+ channels. Although the availability of detailed structural information regarding the channel pore has facilitated the interpretation of data from these channels, the blocker-induced full conductance (complete) block of ionic flux makes it indistinguishable from the normal closed or inactivated events. Therefore, the transitions between the blocked and closed or inactivated states are not directly visible in the single channel data, leading to loss of vital information pertaining to channel gating behavior.The cardiac ryanodine receptor (RyR2) is a massive (∼2.2MDa) Ca2+- activated Ca2+ release channel responsible for transducing the information from an incoming action potential to trigger cardiac contraction. It is a cation selective channel that exhibits a large single channel conductance for K+ (∼723 pS in 210 mM K+) but is only blocked to subconductance states by QA blockers, allowing detailed study of state transitions in the presence of an ongoing block. In this study, the kinetics of single recombinant mouse RyR2 channels reconstituted in planar lipid bilayers were examined in the presence of the QA blockers tetrabutyl (TBA)- and tetrapentyl ammonium (TPeA). Detailed analyses of single channel data and subsequent modeling of gating behavior using Hidden Markov model based algorithms reveals novel effects of blockers which differentially affect gating in the putative selectivity filter and the inner helix bundle crossing regions of the RyR2 pore, and provides further evidence in favor of the possible existence of these two distinct gating entities in the absence of direct evidence from crystal structures. Research supported by British Heart Foundation.

The Stretch-Activated BK (SAKca) Channel in Chick Heart is Inhibited by the Spider Peptide GsMTx-4

GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata and inhibits atrial fibrillation potentiated by dilatation in heart by blocking the stretch-activated (SA) ion channels. However, the mechanism by which GsMTx-4 inhibits SA channels remains unknown. Here we report that the extracellularly applied nM concentrations of GsMTx4 inhibited SAKca channels in excised inside-out patch membranes of isolated chick heart cells. We found that GsMTx4 inhibited the stretch-induced SAKca channel opening in a dose-dependent manner without altering the single-channel conductance. Membrane stretch shifted the voltage dependence of activation of SAKca to more negative voltages, whereas a relatively low concentration of extracellularly applied GsMTx-4 could reverse this effect. Detailed single-channel kinetics analysis revealed that GsMTx-4 could antagonize the increases in the open-time duration induced by membrane stretch. In addition, we found that the sensitivity of SAKca channel towards GsMTx-4 effect was eliminated by the deletion of 59-amino-acid STREX (stress axis regulated exon) located between RCK1 and RCK2 domains in the C-terminus of SAKca channel, suggesting that STREX is the potential target site for GsMTx-4 action in SAKca channel. Our data establish that GsMTx-4 acts as a gating modifier on SAKca channel from chick heart, and may represent a common inhibition mechanism of GsMTx-4 against all types of mechanosensitive (MS) ion channels. Our finding also suggest SA channel is a potential target for curing atrial fibrillation by using GsMTx-4, thus may provide a new insight into the treatment of cardiac arrhythmia.

Single-channel kinetics of BK (Slo1) channels.

Single-channel kinetics has proven a powerful tool to reveal information about the gating mechanisms that control the opening and closing of ion channels. This introductory review focuses on the gating of large conductance Ca2+- and voltage-activated K+ (BK or Slo1) channels at the single-channel level. It starts with single-channel current records and progresses to presentation and analysis of single-channel data and the development of gating mechanisms in terms of discrete state Markov (DSM) models. The DSM models are formulated in terms of the tetrameric modular structure of BK channels, consisting of a central transmembrane pore-gate domain (PGD) attached to four surrounding transmembrane voltage sensing domains (VSD) and a large intracellular cytosolic domain (CTD), also referred to as the gating ring. The modular structure and data analysis shows that the Ca2+ and voltage dependent gating considered separately can each be approximated by 10-state two-tiered models with five closed states on the upper tier and five open states on the lower tier. The modular structure and joint Ca2+ and voltage dependent gating are consistent with a 50 state two-tiered model with 25 closed states on the upper tier and 25 open states on the lower tier. Adding an additional tier of brief closed (flicker states) to the 10-state or 50-state models improved the description of the gating. For fixed experimental conditions a channel would gate in only a subset of the potential number of states. The detected number of states and the correlations between adjacent interval durations are consistent with the tiered models. The examined models can account for the single-channel kinetics and the bursting behavior of gating. Ca2+ and voltage activate BK channels by predominantly increasing the effective opening rate of the channel with a smaller decrease in the effective closing rate. Ca2+ and depolarization thus activate by mainly destabilizing the closed states.

The Effect of Autism Candidate-Gene Mutations in the Voltage-Gated Calcium Channel β2 Subunit on Single Channel Kinetics

The electrophysiological properties of voltage-gated calcium channel (VGCC) complexes are determined by the specific combination of subunit isoforms, i. e. of one pore-forming CaVα1 subunit and the auxiliary subunits β, α2-δ (and γ). Therefore, VGCCs differentially modulate the cellular response to stimuli, which is relevant for neuronal function. Interestingly, the CaVα1C and CaVβ2-subunit genes have been shown to be risk loci for five major psychiatric disorders including autism spectrum disease (ASD) (Lancet (2013) 381:1371). Our group has recently found three missense mutations in the CaVβ2 gene in ASD patients; while two mutants (G167S, S197F) resulted in a retardation of inactivation behavior, one mutant (F240L) accelerated the inactivation of whole-cell Ba2+ currents (PLOS One (2014) 9(4): e95579). In the present study, we performed single channel patch clamp of HEK cells co-transfected with CaVβ2 mutants and CaVα1C. The gating parameters revealed a pronounced biophysical phenotype for all mutations: G167S and S197F persisted longer in an open state by elongating its mean open time (p = 0.005). F240L showed a trend for an increased open probability. Further, the transition rate constants obtained from Markov modelling of the single-channel data were consistent with the observed gating parameters. Here, the Markov model revealed significantly decelerated transition from open to closed state for G167S (p = 0.008) and S197F (p = 0.03). Both G167S and S197F showed slower transition rates suggesting a preference for deeper closed states (for S197F p = 0.04). That would explain why their open probabilities are not increased while mean open times are elongated. We conclude that the three mutations, each exhibiting different biophysical mechanisms, lead to the same outcome: more channel activity.

Quasithermodynamic contributions to the fluctuations of a protein nanopore.

Proteins undergo thermally activated conformational fluctuations among two or more substates, but a quantitative inquiry on their kinetics is persistently challenged by numerous factors, including the complexity and dynamics of various interactions, along with the inability to detect functional substates within a resolvable time scale. Here, we analyzed in detail the current fluctuations of a monomeric β-barrel protein nanopore of known high-resolution X-ray crystal structure. We demonstrated that targeted perturbations of the protein nanopore system, in the form of loop-deletion mutagenesis, accompanying alterations of electrostatic interactions between long extracellular loops, produced modest changes of the differential activation free energies calculated at 25 °C, ΔΔG⧧, in the range near the thermal energy but substantial and correlated modifications of the differential activation enthalpies, ΔΔH⧧, and entropies, ΔΔS⧧. This finding indicates that the local conformational reorganizations of the packing and flexibility of the fluctuating loops lining the central constriction of this protein nanopore were supplemented by changes in the single-channel kinetics. These changes were reflected in the enthalpy–entropy reconversions of the interactions between the loop partners with a compensating temperature, TC, of ∼300 K, and an activation free energy constant of ∼41 kJ/mol. We also determined that temperature has a much greater effect on the energetics of the equilibrium gating fluctuations of a protein nanopore than other environmental parameters, such as the ionic strength of the aqueous phase as well as the applied transmembrane potential, likely due to ample changes in the solvation activation enthalpies. There is no fundamental limitation for applying this approach to other complex, multistate membrane protein systems. Therefore, this methodology has major implications in the area of membrane protein design and dynamics, primarily by revealing a better quantitative assessment on the equilibrium transitions among multiple well-defined and functionally distinct substates of protein channels and pores.

Modulation of CFTR gating by permeant ions

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

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.

Modulation of non-NMDA receptor gating by auxiliary subunits.

During the past decade, considerable evidence has accumulated that non-NMDA glutamate receptors (both AMPA and kainate subtypes) are modulated by the association of the core tetrameric receptor with auxiliary proteins that are integral components of native receptor assemblies. This short review focuses on the effect of two types of auxiliary subunits on the biophysical properties and kinetic behaviour of AMPA and kainate receptors at the level of single receptor molecules. Type I transmembrane AMPA receptor proteins increase the number of AMPA receptor openings that result from a single receptor activation as well as the proportion of openings to conductance levels above 30 pS, resulting in larger peak ensemble currents that decay more slowly and bi-exponentially. Co-expression of Neto1 and 2 with pore-forming kainate receptor subunits also increases the duration of bursts and destabilizes desensitized states, resulting in a rapid component of recovery and clusters of bursts that produce a slow component in desensitization decays. The distinct gating seen in the presence of auxiliary subunits reflects slow switching between gating modes with different single-channel kinetics and open probability. At any given time, the relative proportions of receptors in each gating mode determine both the shape and the amplitude of synaptic currents.

Non-Equilibrium Gating of CFTR Revealed by Nitrate as Charge Carriers

CFTR, a member of the ATP-binding cassette protein superfamily, is a phosphorylation-activated but ATP-gated chloride channel. Like other anion channels, CFTR's pore is permeable to a wide variety of anions, including bromide, nitrate, iodide and bicarbonate. From the shift of reversal potentials under bi-ionic conditions for chloride, bromide and nitrate, we obtained a permeability sequence: NO3- > Br- > Cl-, a result consistent with previous reports, but the macroscopic conductance sequence, NO3- > Br- > Cl-, contradicts previously published Cl- > NO3- > Br-. Nonetheless, single-channel studies reveal a conductance sequence of Cl- > NO3- > Br-, suggesting bromide and nitrate may affect CFTR gating. By analyzing single channel kinetics, we found that NO3- indeed increases the open probability (0.71±0.01 versus 0.51±0.02 with Cl-) by increasing the opening rate and prolonging the open time. Interestingly, when examining recordings from patches containing one single channel in nitrate-based bath, we observed two distinct open-channel conductance levels (the smaller O1 state and the larger O2 state), a phenomenon similar to the effect of the R352C mutation on CFTR. Furthermore, statistical analysis of the pattern of gating transitions also reveals a prevalent transition of C→O1→O2→ C (46.3%) over C→O2→O1→ C (< 5%), a telltale sign for a violation of detailed balance and hence demanding an input of free energy to drive the gating transition in a preferred direction. In addition, a considerable fraction of openings contain more than one O1→O2 transition (38%), supporting the idea that more than one ATP molecule is hydrolyzed within an opening burst. Overall our studies indicate that nitrate, as a charge carrier, can be a new tool to probe CFTR's non-equilibrium gating cycle.

Lessons from KATP Channels with Diabetogenic Mutations in Sulfonylurea Receptor 1 (SUR1)

Numerous mutations have been identified in SUR1 (ABCC8) subunit of the neuroendocrine type KATP channel in subjects with neonatal diabetes, neonatal diabetes plus epilepsy and/or other neurological features, maturity-onset diabetes of young, and later-onset diabetes. Patch-clamping, single-channel kinetics analysis, affinity photolabeling and molecular modeling were used to clarify how diabetogenic mutations in different parts of SUR1 affect open probability and sulfonylurea inhibition of SUR1/Kir6.2 KATP channel. Essentially all tested diabetogenic mutations in the canonical TMD1-NBD1-TMD2-NBD2 ABC exporter core of SUR1 hyperactivated KATP by stabilizing the stimulatory Mg-nucleotide bound (outward facing) state of SUR1 without affecting the intrinsic gating of KATP channel or its sensitivity to inhibitory nucleotides. Hyperstimulated mutant channels showed attenuated sulfonylurea inhibition in the presence, but not the absence, of stimulatory MgATP/ADP, indicating that KATP with SUR1 in the inward facing state has the lowest Kd for sulfonylureas. Diabetogenic mutations in the non-canonical TMD0-L0 part of SUR1 hyperactivated KATP by destabilizing its long-lived closed state with the highest affinity to inhibitory ATP or by strengthening the functional coupling between the MgATP/ADP-bound SUR1 core and the active (burst) state of the Kir pore. Mutations destabilizing the long-lived closed state compromised sulfonylurea inhibition of KATP in the absence of nucleotides but not the drug-induced release of stimulatory nucleotides. The findings support the mechanistic model (FEBS Letters, 585:3555-9) in which the TMD0-L0 module couples the SUR1 core with the KATP pore, define the most common ABCC8-associated mechanisms of KATP hyperactivity, and largely explain why the majority of diabetic subjects with mutant SUR1 require body-weight normalized doses of sulfonylureas exceeding those recommended by the FDA for treatment of common type 2 diabetes.

A neurosteroid potentiation site can be moved among GABAA receptor subunits

Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid.

Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.

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.