Subconductance States Research Articles

Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Gating mechanism of mechanosensitive channel of large conductance: a coupled continuum mechanical-continuum solvation approach.

Gating transition of the mechanosensitive channel of large conductance (MscL) represents a good example of important biological processes that are difficult to describe using atomistic simulations due to the large (submicron) length scale and long (millisecond) time scale. Here we develop a novel computational framework that tightly couples continuum mechanics with continuum solvation models to study the detailed gating behavior of E. coli-MscL. The components of protein molecules are modeled by continuum elements that properly describe their shape, material properties and physicochemical features (e.g., charge distribution). The lipid membrane is modeled as a three-layer material in which the lipid head group and tail regions are treated separately, taking into account the fact that fluidic lipid bilayers do not bear shear stress. Coupling between mechanical and chemical responses of the channel is realized by an iterative integration of continuum mechanics (CM) modeling and continuum solvation (CS) computation. Compared to previous continuum mechanics studies, the present model is capable of capturing the most essential features of the gating process in a much more realistic fashion: due mainly to the apolar solvation contribution, the membrane tension for full opening of MscL is reduced substantially to the experimental measured range. Moreover, the pore size stabilizes constantly during gating because of the intricate interactions of the multiple components of the system, implying the mechanism for sub-conducting states of MscL gating. A significant fraction ([Formula: see text]2/3) of the gating membrane strain is required to reach the first sub-conducting state of our model, which is featured with a relative conductance of 0.115 to the fully opened state. These trends agree well with experimental observations. We anticipate that the coupled CM/CS modeling framework is uniquely suited for the analysis of many biomolecules and their assemblies under external mechanical stimuli.

Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

Resolving Active Ion Transport at the Single Molecule Level for the First Time

Electrochemical gradients across cellular membranes control a plethora of vital biological processes. These gradients are generated by primary active transporters and are subsequently used to drive the exchange of other solutes through secondary active transporters and to facilitate signaling via ion channels. The macroscopic biological phenomena that channels and transporters give rise to are intimately connected to how they function at the single molecule level. For decades, patch clamp recording has been used to observe the functional dynamics of single ion channels revealing discrete on and off states, subconductance states, and other mechanistically important features that macroscopic experiments cannot probe(1). Currently, there aren’t any techniques available to investigate transporter function at the single molecule level, thus they are only studied using ensemble biochemical methods.For a decade we have been developing quantitative fluorescence microscopy based assays of arrayed proteoliposomes for investigating membrane proteins(2-6). Here we extended the platform to monitor the single-molecule activity and the regulation of a prototypic P-type ATPase, Arabidopsis thaliana H+-ATPase (AHA2). For the first time we have shown that individual proton pumps are not active continuously but rather transitioning between active and inactive states separated by a large activation barrier (kJMole−1). We found that the dynamics of these states form the basis of the regulation of the macroscopic activity either by regulatory R-domain, pH gradients, or ATP. Like for ion channels we often found that regulatory inputs do not affect the intrinsic pumping rates but rather active probabilities.

Molecular Determinants Underlying the Pathogenic Mechanism of Kid Syndrome Elicited by Cx26G12R Mutation

Several mutations in Cx26 promote keratitis ichthyosis deafness (KID), a rare disease in which patients exhibit deafness, skin and corneal disorders. Some KID mutations are located in the amino terminal segment of Cx26, which has been proposed as the putative voltage sensor. Most syndromic mutations elicit a gain of hemichannel function, however the mechanism underlying this phenomenon remains unclear. Here we examined the biophysical properties of the mutant G12R at macroscopic and single channel levels. We perform two-electrode voltage clamp to study the deactivation kinetics and apparent affinity for calcium of hemichannel currents. In contrast to wild-type (wt) Cx26, we found that the activation kinetics of the mutant lack saturation of the current upon depolarization pulses above +40 mV. Estimates of the deactivation time constant yield values of 5s and 10s for G12R and wtCx26 hemichannels, respectively; however the apparent affinity for calcium remains similar to wtCx26. Single channel recordings analysis showed that the mutant virtually eliminates the transitions to the subconductance state. Moreover, although the single channel conductance was similar to wtCx26 hemichannels (320 pS), the open probability and mean open times of G12R hemichannels become larger. Molecular dynamics indicates that the G12R induce an electrostatic effect and ddG calculations support protein destabilization.Supported by Fondecyt-3150634 to IEG; Fondecyt-3140590 to GC; Fondecyt-1130855 to ADM; Anillo-ACT1104 to ADM and CG; Fondecyt-1120802 to CG; Grant RO1-GM099490 to J.E.C; AP has a Conicyt Fellowship. The Centro Interdisciplinario de Neurociencias de Valparaiso is a Chilean Millennium Institute (P09-022-F).

Structural Insights into Ryanodine Receptor-FKBP12 Interactions in Wild-Type and Mutated States

Ryanodine receptors (RyRs) control the release of intracellular calcium from sarcoplasmic reticulum and play a central role in excitation-contraction coupling of skeletal and cardiac muscle. Many modulatory proteins and small molecules interact and regulate the function of RyR, including FKBP12 (FK506 binding proteins of 12), a protein ligand that binds RyR with nanomolar affinity. FKBP12 stabilizes the closed state of the channel and reduces the appearance of subconductance states. Dissociation of FKBP12 can cause the calcium leak from RyR and has been implicated in several disorders, such as muscular dystrophy, sarcopenia, heart failure, diabetes, or Alzheimer's. Previous studies using cryo-EM and FRET suggest FKBP12 binds to the periphery of cytoplasmic domain, but the exact binding determinants within the RyR protein at the single amino acid level had remained unclear. The recent high-resolution cryo-EM studies also disagree on the identity of the RyR domain interacting with FKBP12. We determined the high-resolution crystal structures of several RyR domains. Computational docking of these structures, combined with FRET trilateration experiments, show that SPRY1 domain is located next to FKBP12. The key residues in SPRY1 critical for the binding of FKBP12 were identified by molecular dynamics flexible fitting and verified by mutagenesis and functional assays. Disease mutations in SPRY1 were investigated by x-ray crystallography. One of them directly affects FKBP12 binding properties by interfering domain folding. This structural model provides a valuable template for designing therapeutic drugs targeting RyR-FKBP12 interface to treat the diseases mentioned above.

Distinct Pores for Peroxisomal Import of PTS1 and PTS2 Proteins.

Two peroxisomal targeting signals, PTS1 and PTS2, recognized by cytosolic receptors Pex5 and cooperating Pex7/Pex18, direct folded proteins to the peroxisomal matrix. A pore consisting of the PTS1 receptor Pex5 and the docking protein Pex14 imports PTS1 proteins. We identified a distinct PTS2-specific pore, which contains the PTS2 co-receptor Pex18 and the Pex14/Pex17-docking complex as major constituents. The estimated maximal pore size of ∼ 4.7 nm is large enough to allow import of folded PTS2 proteins. PTS2 cargo proteins modulate complex gating, open probability, and subconductance states of the pore. While the PTS1 channel is transiently activated by arriving receptor-cargo complexes, the reconstituted PTS2 channel is constitutively present in an open state. However, the cargo-loaded PTS2 channel is largely impermeable to solutes and ions. Our results demonstrate that import of PTS1 and PTS2 proteins does not converge at the peroxisomal membrane as previously anticipated but is performed by distinct pores.

Subconductance Gating and Voltage Sensitivity of Sarcoplasmic Reticulum K+ Channels: A Modeling Approach

Sarcoplasmic reticulum (SR) K+ channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K+ channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K+ channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K+ channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K+ channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K+ flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.

The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.

Functional characterization of sticholysin I and W111C mutant reveals the sequence of the actinoporin's pore assembly.

The use of pore-forming toxins in the construction of immunotoxins against tumour cells is an alternative for cancer therapy. In this protein family one of the most potent toxins are the actinoporins, cytolysins from sea anemones. We work on the construction of tumour proteinase-activated immunotoxins using sticholysin I (StI), an actinoporin isolated from the sea anemone Stichodactyla helianthus. To accomplish this objective, recombinant StI (StIr) with a mutation in the membrane binding region has been employed. In this work, it was evaluated the impact of mutating tryptophan 111 to cysteine on the toxin pore forming capability. StI W111C is still able to permeabilize erythrocytes and liposomes, but at ten-fold higher concentration than StI. This is due to its lower affinity for the membrane, which corroborates the importance of residue 111 for the binding of actinoporins to the lipid bilayer. In agreement, other functional characteristics not directly associated to the binding, are essentially the same for both variants, that is, pores have oligomeric structures with similar radii, conductance, cation-selectivity, and instantaneous current-voltage behavior. In addition, this work provides experimental evidence sustaining the toroidal protein-lipid actinoporins lytic structures, since the toxins provoke the trans-bilayer movement (flip–flop) of a pyrene-labeled analogue of phosphatidylcholine in liposomes, indicating the existence of continuity between the outer and the inner membrane leaflet. Finally, our planar lipid membranes results have also contributed to a better understanding of the actinoporin’s pore assembly mechanism. After the toxin binding and the N-terminal insertion in the lipid membrane, the pore assembly occurs by passing through different transient sub-conductance states. These states, usually 3 or 4, are due to the successive incorporation of N-terminal α-helices and lipid heads to the growing pores until a stable toroidal oligomeric structure is formed, which is mainly tetrameric.

Mechanosensitive unpaired innexin channels in C. elegans touch neurons.

Invertebrate innexin proteins share sequence homology with vertebrate pannexins and general membrane topology with both pannexins and connexins. While connexins form gap junctions that mediate intercellular communication, pannexins are thought to function exclusively as plasma membrane channels permeable to both ions and small molecules. Undoubtedly, certain innexins function as gap junction proteins. However, due to sequence similarity to pannexins, it was postulated that innexins also function as plasma membrane channels. Indeed, some of the leech innexins were found to mediate ATP release as unpaired membrane channels with shared pharmacology to pannexin channels. We show here that Caenorhabditis elegans touch-sensing neurons express a mechanically gated innexin channel with a conductance of ∼1 nS and voltage-dependent and K(+)-selective subconductance state. We also show that C. elegans touch neurons take up ethidium bromide through a mechanism that is activated and blocked by innexin activating stimuli and inhibitors, respectively. Finally, we present evidence that touch neurons' innexins are required for cell death induced by chemical ischemia. Our work demonstrates that innexins function as plasma membrane channels in native C. elegans neurons, where they may play a role in pathological cell death.

What Makes You Can also Break You, Part III: Mitochondrial Permeability Transition Pore Formation by an Uncoupling Channel within the C-Subunit Ring of the F1FO ATP Synthase?

What Makes You Can also Break You, Part III: Mitochondrial Permeability Transition Pore Formation by an Uncoupling Channel within the C-Subunit Ring of the F1FO ATP Synthase?

Characterization of ryanodine receptor type 1 single channel activity using “on-nucleus” patch clamp

In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca2+ release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca2+] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca2+, and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

La liberación de calcio de los depósitos intracelulares promueve la secreción de serotonina en terminales sinápticas

En este trabajo se estudio la participación que tiene la liberación de calcio del retículo endoplásmico en la liberación de serotonina en terminales sinápticas. Los experimentos se llevaron a cabo en sinapsis formadas en cultivo entre neuronas serotonergicas de Retzius y neuronas mecanosensoriales sensibles a presión, aisladas del Sistema Nervioso Central de la sanguijuela. En esta preparación la estimulación con pares de impulsos produjo facilitación sináptica. La estabilización de los receptores de rianodina en un estado de sub-conductancia por la incubación con rianodina 100 µM produjo un alargamiento del potencial sináptico en respuesta a impulsos presinápticos, sugiriendo que el calcio liberado por estos canales puede alcanzar las vesículas y promover la secreción. En contraste, el vaciamiento de los depósitos intracelulares de calcio con tapsigargina 500 nM produjo una disminución gradual de la facilitación sináptica ante impulsos presinápticos pareados y abolió la liberación extrasináptica en el axón neuronal en respuesta a trenes de impulsos. Todo esto ocurrió sin cambios en las propiedades de la membrana postsináptica, lo cual sugiere que la liberación de calcio intracelular participa en un mecanismo de retroalimentación positiva que promueve la liberación presináptica y perisináptica en las neuronas serotonérgicas.

Plant VDAC Selectivity and Voltage-Dependence are Uncoupled

The mitochondrial respiration requires the exchange of inorganic ions and metabolites between the cytoplasm and the mitochondrial matrix. This entails the translocation of these solutes through both inner and outer mitochondrial membranes. This function is fulfilled by the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane. A typical feature of VDAC is its voltage-dependence. At low voltage, typically |V|<20mV, it is in its open state and it switches to subconductance states upon increasing the voltage amplitude. It is generally assumed that both selectivity and voltage-dependence of VDAC are coupled: the VDAC is anion selective in the open state but cation selective in subconducance states. However, it was shown that the selectivity of the open state can spontaneously switch between anion and cation selectivity [1]. The origin of this selectivity inversion is not known. We studied the selectivity of the plant PcVDAC32 that belong to the canonical isoform of VDAC including mammalian VDAC1 and yeast VDAC1 with which it shares similar electrophysiological properties as well as secondary structure content [2]. We show that change of selectivity occurs in open and in subconductance states without being related to gating. In the open state the probability of selectivity inversion is regulated by the membrane lipid composition, the ion strength and the magnitude of the ion gradient across the membrane. Furthermore no matter the experimental condition the selectivity inversion is not correlated to a change in channel conductance. Altogether these results indicate that there is no correlation between the VDAC selectivity and its voltage-dependence.[1] Pavlov, E. et al. (2005) BBA 1710: 96.[2] Homble, F., Krammer,E.V. and Prevost, M. (2012) BBA 1818: 1486.This work was supported by the FRS-FNRS (Belgium).

Coupled Gating of Ryanodine Receptors: Evidence for a Role of Physical RyR-RyR Interactions

We studied the synchronous function (coupled gating) of arrays of striated muscle ryanodine receptors (RyRs) reconstituted into planar bilayers. We think that understanding coupled gating would help explain local Ca2+ sparks and global Ca2+ transients which arise from the simultaneous activation of groups of RyRs in the sarcoplasmic reticulum. We have previously found that coupled gating of multiple cardiac or skeletal RyRs requires luminal Ca2+ as current carrier. In principle, this would suggest that Ca2+ flowing from the open pore of a RyR, could activate neighboring channels (local CICR). However, further analysis showed that coupled events are insensitive to changes in the magnitude of lumen-to-cytosol Ca2+ flux. This is not compatible with the idea of individual channels modulated by local CICR. As an alternative, we tested the involvement of physical RyR-RyR interactions in the process of coupled gating. We found that coupled gating of RyRs reconstituted in planar bilayers is not significantly affected by agents that disrupt cytoskeletal networks, FKBPs and/or kinases/phosphatases. However, the polycationic amine spermine interfered with coupled RyR gating (suggesting a role for electrostatic interactions). Additionally ryanodol, known to lock open channels in a sub-conductance state, also affected coupled RyRs. Nevertheless, event termination (synchronous channel closures) occurred even during ryanodol substates. This was unexpected because single channels do not transition from the ryanodol/ryanodine substate to the closed state. Overall, our results suggest that modulation of coupled RyRs is different from that expected for individual channels interacting by local CICR. We think that luminal Ca2+ promotes conformational changes in RyRs allowing for cytosolic RyR-RyR interactions of electrostatic nature that induce coupled events (Supported by NIH R01 GM078665 and AHA-MWA 12180038).

Phosphorylation of Maurocalcine Strongly Modifies its Effect on Type 1 Ryanodine Receptor

Maurocalcine (MCa) is a 33-amino acid peptide isolated from the venom of Scorpio maurus palmatus, a Tunisian scorpion. It possesses efficient cell penetration properties and has been shown to strongly modify ryanodine receptor (RyR1) channel behavior by increasing open probability and inducing a long-lasting subconductance state. The amino acid residue threonine at position 26 belongs to a putative phosphorylation site within MCa sequence. We investigated the effect of a) T26 phosphorylation (MCa T26Phospho) by protein kinase A and b) replacement of T26 by glutamic acid residue to mimic phosphorylation on the effect of MCa on RyR1 properties. Using [3H]ryanodine ([3H]Ry) binding and single channel voltage-clamp measurements, we show that both MCa T26Phospho and MCa T26E analogs almost completely lose the ability to induce RyR1 activation. Neither MCa T26Phospho nor MCa T26E (up to 2µM) increase [3H]rya binding. Small increase (∼1.65-fold activation; p<0.01) was observed with high concentrations (10µM) of either MCa T26Phospho or MCa T26E. Single channel measurements revealed that neither MCa T26E nor MCa T26Phospho (up to 2µM) were able to induce the characteristic long-lasting subconductance state of RyR1 observed in presence of wild-type MCa. MCa T26Phospho altered RyR1 channel gating in a time-dependent biphasic manner with initial activation of Po followed by almost complete inactivation. These effects of MCa T26Phospho could be reversed by 200nM wt MCa. At concentrations ≥2μM, MCa T26E enhances Po∼3 fold by shortening τc without altering τo. These results show that single phosphorylation of MCa modifies RyR1 activity.1P01 AR52354.

Diminazene Interaction with ASIC1A Channels

Diaryldiamidines, a class of DNA minor groove binders used clinically since the 1940s in the treatment of tropical parasitoses such as trypanosomiasis, leishmaniasis, pneumocystis pneumonia and babesiosis, have been recently found to exert specific blocking effects on acid-sensing ion channels (ASIC), raising hopes to develop new small molecule neuroprotective agents for ischemic stroke, which involves acidosis-mediated activation of ASIC1a and heteromeric ASIC1a/2b channels. Therefore we have thoroughly investigated the modes of interaction of diminazene with ASIC1a via whole-cell and single-channel recordings on outside-out patches excised from HEK293 cells of ASIC1a transient currents evoked by brief acidic pulses (pH 6.0). Data idealization and model-dependent fitting were performed with custom hidden Markov model maximum likelihood algorithms. We proved that the 3 subconductance levels are due to partial block by calcium of the permeation pathway, and expanded our previous 8-state linear connectivity gating model (Marin et al. 2008 Channels 2(6):1-10) including 3 subconductance states for each of the 2 gating modes. Single-channel recordings with diminazene 0.6 or 3 μM indicated a mechanism of open channel block. By exponential fits of the distributions of blocked and unblocked periods for each gating mode we computed binding and unbinding rates and dissociation constants of 0.13 and 0.27 μM for gating modes 2 and 1, respectively. Using atomic resolution models of human ASIC1a in the open conformations corresponding to the 2 gating modes, we found via molecular docking several intra- and intersubunit binding sites for diminazene. One of these sites, assumed to be responsible for the state-dependent block, features protrusion of the drug in the transmembrane domain permeation pathway. As expected, for this blocking site the interaction energy is higher in the symmetrical open conformation of the second gating mode than in the asymmetrical open conformation of the first gating mode.

H+ Inhibits TRIC-B Channels Derived from Mouse TRIC-A Knockout Tissue

Trimeric intracellular cation channels (TRIC-A/TRIC-B) are found in the endo/sarcoplasmic reticulum (ER/SR) and nuclear membranes. TRIC-B knockout mice die in respiratory failure following birth but there are additional diverse, severe pathological symptoms (Yamazaki D. et al. (2009)). The physiological roles of TRIC-B are not fully understood and few regulators of channel activity are known. We here investigate if TRIC-B function is sensitive to pH by incorporating light SR membrane vesicles from TRIC-A knockout skeletal muscle into artificial membranes under voltage-clamp conditions in symmetrical 210 mM K-PIPES, pH 7.2. Open probability was determined at ±30 mV when ≤2 channels were gating in the bilayer. With ≥3 channels present, noise analysis was performed because the complex sub-conductance state gating prevented accurate measurements. As previously observed (Venturi E. et al. (2009)), TRIC-B was more open at positive potentials but exhibited variable gating behaviour. For example, at pH 7.2, at +30 mV, noise analysis yielded a mean current of 1.25±0.25 pA (SEM; n=23) but ranged from 0.025 to 3.36 pA. We observed that TRIC-B became more open as cytosolic pH was increased. At +30 mV, as cytosolic pH was raised from 7.2 to 9.2, we observed a 3.34 fold increase in mean current (SEM; n=7; p<0.05) whereas mean current decreased 3.33 fold when cytosolic pH was lowered from pH 7.2 to 6.2, A similar trend was observed when luminal pH was altered. Under acidic conditions where both cytosolic and luminal pH was lowered, TRIC-B channel opening was markedly inhibited and the effects of cytosolic and luminal [H+] appeared to be additive.Further experimentation is required to understand the mechanisms underlying pH regulation of TRIC-B function and the physiological/pathophysiological significance of TRIC-B sensitivity to pH.Supported by the BHF, EPSRC and JSPS.

Structure-Function Relationship of Calcins, a Family of High-Affinity Peptide Ligands of Ryanodine Receptors

Calcins is a novel family of scorpion peptides that bind with high affinity and specificity to ryanodine receptors (RyRs) and increase their open probability by inducing the appearance of a long-lasting subconductance state. Here we report two newly-identified calcins (Vejocalcin and Urocalcin) and provide a comprehensive analysis of the structure-function relationship of the eight calcins known to date, based on primary sequence examination, 3D structure modeling, and their effect on RyR1. [3H]Ryanodine binding assays, used as index of the open probability (Po) of RyRs, revealed that all eight calcins (Opicalcin1, Opicalcin2, Imperacalcin, Maurocalcin, Hadrucalcin, Hemicalcin, Vejocalcin, and Urocalcin) activate RyR1 dose-dependently, with EC50s (in nM) 0.35, 5, 8, 12, 37, 71, 91, and 523, respectively. At 1 µM, calcins significantly augmented the bell-shaped [Ca2+]-activity curve of RyR1 with potency Opicalcin1>Opicalcin2>Vejocalcin>Urocalcin. In single channel recordings, the heretofore uncharacterized calcins Opicalcin1, Opicalcin2 and Vejocalcin increased the Po of RyR1 significantly and the fractional conductance was ∼0.45, 0.30, and 0.65, respectively, of the full conductance state. Opicalcin1, Maurocalcin, Hadrucalcin and Vejocalcin induced Ca2+ release from rabbit skeletal SR with ED50s (in nM) 2.8±0.02, 8.3±0.25, 16±0.4, 36±0.5, respectively. A clear Ca2+ release-[3H]ryanodine binding activity correlation (r2=0.99) was obtained. Primary sequence alignment and evolutionary analysis (ClustalW and MEGA5.2) showed high homology among all calcins. An inhibitor cysteine-knot (ICK) motif with βαββ domains stabilized by three disulfide bridges characterizes this peptide family, although Vejocalcin lacks the β2 sheet. Positively-charged residue mutations in the high variable N-terminal region (G1-N14) and negatively-charged residue variations in the conserved C-terminal region (D15-R33) greatly decrease the effect of calcins on RyR1. In conclusion, natural variation in calcin peptides offers a diversified set of RyR ligands with capacity to modulate RyRs with high dynamic range and potency.