Single Ca Channel Research Articles

Efficient Approximations for Stationary Single-Channel Ca2+ Nanodomains across Length Scales

We consider the stationary solution for the Ca2+ concentration near a point Ca2+ source describing a single-channel Ca2+ nanodomain in the presence of a single mobile Ca2+ buffer with 1:1 Ca2+ binding. We present computationally efficient approximants that estimate stationary single-channel Ca2+ nanodomains with great accuracy in broad regions of parameter space. The presented approximants have a functional form that combines rational and exponential functions, which is similar to that of the well-known excess buffer approximation and the linear approximation but with parameters estimated using two novel, to our knowledge, methods. One of the methods involves interpolation between the short-range Taylor series of the free buffer concentration and its long-range asymptotic series in inverse powers of distance from the channel. Although this method has already been used to find Padé (rational-function) approximants to single-channel Ca2+ and buffer concentrations, extending this method to interpolants combining exponential and rational functions improves accuracy in a significant fraction of the relevant parameter space. A second method is based on the variational approach and involves a global minimization of an appropriate functional with respect to parameters of the chosen approximations. An extensive parameter-sensitivity analysis is presented, comparing these two methods with previously developed approximants. Apart from increased accuracy, the strength of these approximants is that they can be extended to more realistic buffers with multiple binding sites characterized by cooperative Ca2+ binding, such as calmodulin and calretinin.

Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock

Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.

Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+ Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established. We recently developed a cell-free, in vitro biochemical assay, termed SV pull-down (SV-PD), to test for SV binding proteins and used this to demonstrate that CaV2.2 or the distal third of its C-terminal can capture SVs. In subsequent reports we identified the binding site and characterized an SV binding motif. In this study, we set out to test if a similar SV-binding mechanism exists in the primary presynaptic channel type, CaV2.1. We cloned the chick variant of this channel and to our surprise found that it lacked the terminal third of the C-terminal, ruling out direct correlation with CaV2.2. We used SV-PD to identify an SV binding site in the distal half of the CaV2.1 C-terminal, a region that corresponds to the central third of the CaV2.2 C-terminal. Mutant fusion proteins combined with motif-blocking peptide strategies identified two domains that could account for SV binding; one in an alternatively spliced region (E44) and a second more distal site. Our findings provide a molecular basis for CaV2.1 SV binding that can account for recent evidence of C-terminal-dependent transmitter release modulation and that may contribute to SV tethering within the CaV2.1 single channel Ca2+ domain.

Subcellular Ca2+ Puffs Mediated By Different Inositol Trisphosphate Receptor Isoforms

The second messenger inositol trisphosphate (IP3) evokes Ca2+ liberation through IP3 receptor/channels (IP3Rs) in the ER membrane to generate a hierarchy of cytosolic signals, including local Ca2+ puffs that arise from concerted openings of clustered IP3Rs, and global Ca2+ waves that propagate through the cell. Mammalian cells express three isoforms of IP3Rs that are encoded by different genes, have ~ 60–80% sequence homology and are reported to display differing functional characteristics. Imaging Ca2+ puffs with single‐channel resolution provides important information on the localization and properties of IP3Rs in intact cells. However, interpretation has been complicated because studies have been limited to native cell types that typically express varying proportions of two or three IP3R isoforms, with additional complexity arising from hetero‐multimerization within the tetrameric IP3R. Here, we applied high‐resolution TIRF microscopy to image Ca2+ puffs in HEK‐293 cell lines in which IP3R isoforms were knocked‐out in pairs by CRISPR/Cas9 technology to generate cells expressing exclusively IP3R types 1, 2 or 3. Puffs were evoked by photoreleased i‐IP3 in all three cell lines, indicating that IP3Rs of each isoform are able to assemble into the clusters that underlie these local Ca2+ signals. Moreover, Ca2+ puffs mediated by all three isoforms displayed largely similar mean amplitudes, temporal characteristics and spatial extents, except that type 3 IP3R‐mediated puffs showed appreciably faster kinetics. A quantal analysis of fluorescence signals revealed the single‐channel Ca2+ flux to be equivalent between isoforms, further suggesting that clusters of different IP3R isoforms contain similar numbers of active channels. Finally, mapping the distances between puff centroid localizations suggested that clusters of type 1 IP3Rs are more dispersed than clusters of type 2 or 3 IP3Rs. Overall, our results indicate that, although there is substantial functional redundancy among the IP3R isoforms, each exhibit distinct properties and spatial arrangements that likely shape the spatial‐temporal patterning of cellular Ca2+ signals observed in native cells.Support or Funding InformationThis work was supported by NIH grants R01 DE019245 (D.I.Y) and R37 GM048071 (I.P)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contribution of BKCa channel to tone regulation by PDE3 and PDE4 depends on the mode of cAMP stimulation and is lost in heart failure

Regulation of vascular tone by 3’,5’-cyclic adenosine monophosphate (cAMP) involves many effectors, including large conductance, Ca 2+ -activated, K + (BK Ca ) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3 and PDE4). Here, we examined the specific contribution of BK Ca channels to tone regulation by these PDEs in rat coronary arteries (CA), and how it may be altered in heart failure (HF). CA were isolated from young adult rats or rats with HF sacrificed 22 weeks after surgical stenosis of the ascending aorta. Age-matched sham animals were used as controls. Tension measurements were conducted on CA contracted using the thromboxan analogue U46619. Myocytes were isolated from CA and used for patch clamp recordings or for proximity ligation assay (PLA). Selective PDE3 (cilostamide, Cil) or PDE4 (Ro-20-1724, Ro) inhibition evoked vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BK Ca channel blocker. Ro and Cil potentiated the relaxation induced by the β-adrenergic agonist isorenaline or the adenylyl cyclase activator L-858051. IBTX abolished the effect of PDE inhibitors on isoprenaline but did not on L-858051. Ro and Cil together increased single BK Ca channel activity in myocytes. CA isolated from rats displaying robust signs of HF showed poor contractile capacity and relaxation to acetylcholine compared to sham rats. In contrast to sham, relaxations induced by PDE inhibitors in HF-CA were not sensitive to IBTX. Expression of the BK Ca channel was 2-fold lower in HF-CA compared to sham-CA. Amount of a 70kDa-PDE4B was increased in HF. PLA demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel. BK Ca -PDE4B duplex was less abundant in HF. BK Ca channels mediate the relaxation of rat CA induced by PDE3 and PDE4 inhibition. Their relative contribution, however, vary with the grade of cAMP stimulation and the physiopathological context of HF.

Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Autosomal-dominant polycystic kidney disease (ADPKD) is a devastating disorder that is characterized by a progressive decline in renal function as a result of the development of fluid-filled cysts. Defective flow-mediated [Ca2+]i responses and disrupted [Ca2+]i homeostasis have been repeatedly associated with cyst progression in ADPKD. We have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) channel is imperative for flow-mediated [Ca2+]i responses in murine distal renal tubule cells. To determine whether compromised TRPV4 function contributes to aberrant Ca2+ regulation in ADPKD, we assessed TRPV4 function in primary cells that were cultured from ADPKD and normal human kidneys (NHKs). Single-channel TRPV4 activity and TRPV4-dependent Ca2+ influxes were drastically reduced in ADPKD cells, which correlated with distorted [Ca2+]i signaling. Whereas total TRPV4 protein levels were comparable in NHK and ADPKD cells, we detected a marked decrease in TRPV4 glycosylation in ADPKD cells. Tunicamycin-induced deglycosylation inhibited TRPV4 activity and compromised [Ca2+]i signaling in NHK cells. Overall, we demonstrate that TRPV4 glycosylation and channel activity are diminished in human ADPKD cells compared with NHK cells, and that this contributes significantly to the distorted [Ca2+]i dynamics. We propose that TRPV4 stimulation may be beneficial for restoring [Ca2+]i homeostasis in cyst cells, thereby interfering with ADPKD progression.-Tomilin, V., Reif, G. A., Zaika, O., Wallace, D. P., Pochynyuk, O. Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Single channel recording of a mitochondrial calcium uniporter

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria.

Novel Approximation of a Stationary Single-Channel Ca2+ Nanodomain

We consider the stationary solution for the Ca2+ concentration near a point Ca2+ source describing a single-channel Ca2+ nanodomain, in the presence of a single mobile buffer with one-to-one Ca2+ binding. Previously, a number of Ca2+ nanodomains approximations have been developed, such as the Excess Buffer approximation, the Rapid Buffering approximation, and the Linear approximation, each valid for appropriate buffering conditions. Apart from providing a simple method of estimating Ca2+ and buffer concentrations without resorting to computationally expensive numerical solution of reaction-diffusion equations, such approximations proved useful in revealing the dependence of nanodomain Ca2+ distribution on crucial parameters such as buffer mobility and its Ca2+ binding properties. Here we present a novel form of analytic approximation, which is based on matching the short-range Taylor series of the nanodomain concentration with the long-range asymptotic series expressed in inverse powers of distance from channel location. Namely, we use a “dual” Padé rational function approximation to simultaneously match terms in the short- and the long-range series, and show that this provides an accurate approximation to the nanodomain Ca2+ and buffer concentrations. We compare this approximation with the previously obtained approximations, and show that it yields a better estimate of the free buffer concentration for a wide range of buffering conditions. The drawback of the presented method is that it has a complex algebraic form above the lowest, bilinear order, and cannot be readily extended to multiple Ca2+ channels. However, it may be possible to extend at least some of the features of this method to the case of cooperative Ca2+ buffers with two Ca2+ binding sites (e.g. calretinin), the case which existing analytic methods do not address. This work was supported in part by the National Science Foundation grant DMS-1517085.

Padé Approximation of a Stationary Single-Channel Ca2+ Nanodomain

We consider the stationary solution for the Ca2+ concentration near a point Ca2+ source describing a single-channel Ca2+ nanodomain, in the presence of a single mobile buffer with one-to-one Ca2+ binding stoichiometry. Previously, a number of Ca2+ nanodomains approximations have been developed, for instance the excess buffer approximation (EBA), the rapid buffering approximation (RBA), and the linear approximation (LIN), each valid for appropriate buffering conditions. Apart from providing a simple method of estimating Ca2+ and buffer concentrations without resorting to computationally expensive numerical solution of reaction-diffusion equations, such approximations proved useful in revealing the dependence of nanodomain Ca2+ distribution on crucial parameters such as buffer mobility and its Ca2+ binding properties. In this study, we present a different form of analytic approximation, which is based on matching the short-range Taylor series of the nanodomain concentration with the long-range asymptotic series expressed in inverse powers of distance from channel location. Namely, we use a “dual” Padé rational function approximation to simultaneously match terms in the short- and the long-range series, and we show that this provides an accurate approximation to the nanodomain Ca2+ and buffer concentrations. We compare this approximation with the previously obtained approximations and show that it yields a better estimate of the free buffer concentration for a wide range of buffering conditions. The drawback of our method is that it has a complex algebraic form for any order higher than the lowest bilinear order, and cannot be readily extended to multiple Ca2+ channels. However, it may be possible to extend the Padé method to estimate Ca2+ nanodomains in the presence of cooperative Ca2+ buffers with two Ca2+ binding sites, the case that existing methods do not address.

Mode switching of Inositol 1,4,5-trisphosphate receptor channel shapes the Spatiotemporal scales of Ca2+ signals.

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel is crucial for the generation and modulation of highly specific intracellular Ca2+ signals performing numerous functions in animal cells. However, the single channel behavior during Ca2+ signals of different spatiotemporal scales is not well understood. To elucidate the correlation between the gating dynamics of single InsP3Rs and spatiotemporal Ca2+ patterns, we simulate a cluster of InsP3Rs under varying ligand concentrations and extract comprehensive gating statistics of all channels during events of different sizes and durations. Our results show that channels gating predominantly in the low activity mode with negligible occupancy of intermediate and high modes leads to single channel Ca2+ release event blips. Increasing occupancies of intermediate and high modes results in events with increasing size. When the channel has more than 50% probability of gating in the intermediate and high modes, the cluster generates very large puffs that would most likely result in global Ca2+ signals. The size, duration and frequency of Ca2+ signals all increase linearly with the total probability of channel gating in the intermediate and high modes. To our knowledge, this is the first study that quantitatively relates the modal characteristics of InsP3R to the shaping of different spatiotemporal scales of Ca2+ signals.

A novel method for approximating equilibrium single-channel Ca2+ domains

Localized calcium (Ca2+) signals control some of the most fundamental physiological processes, including synaptic transmission as well as its activity-dependent plasticity. Computational and mathematical modeling played a crucial role in the understanding of spatio-temporal Ca2+ dynamics that drives these processes, and showed that Ca2+ concentration around a single Ca2+ channel reaches a quasi-stationary distribution (known as the Ca2+ nanodomain) within tens of microseconds after the opening of the channel, and collapses as rapidly after the closing of the channel. Such localization of Ca2+ in time and space is achieved by its rapid diffusion as well as its binding to its multiple interaction partners collectively called Ca2+ buffers and Ca2+ sensors. One of the successes of mathematical modeling was the development of several analytic approximations describing the equilibrium concentration of Ca2+ as a function of distance from the open Ca2+ channel, such as the Rapid Buffering Approximation (RBA), the Linear Approximation (LA) and the Excess Buffering Approximation (EBA) [1-4]. Each of these approximations has a particular applicability parameter regime created by the interplay between the properties of Ca2+ buffers, in particular their mobility and Ca2+ binding rates, and the strength of the Ca2+ current. Here we present a novel approximation method which does not rely on a specific range of the relevant Ca2+ and buffer parameters, and is based on matching the low-distance and large-distance asymptotic behavior of the concentration function. Even at low orders, the resulting approximation is as accurate as the second-order RBA and EBA approximations [4], but its validity extends far beyond the parameter range of applicability of RBA and EBA. The usefulness of the resulting approximation is two-fold: first, together with the previously developed approximations, the novel method could provide a deeper intuition into the dependence of Ca2+ nanodomain properties on the relevant buffering parameters, and second, it constitutes an efficient numerical approximation tool in the modeling of the Ca2+ signals underlying presynaptic and postsynaptic phenomena.

Elementary properties of Ca(2+) channels and their influence on multivesicular release and phase-locking at auditory hair cell ribbon synapses.

Voltage-gated calcium (Cav1.3) channels in mammalian inner hair cells (IHCs) open in response to sound and the resulting Ca2+ entry triggers the release of the neurotransmitter glutamate onto afferent terminals. At low to mid sound frequencies cell depolarization follows the sound sinusoid and pulses of transmitter release from the hair cell generate excitatory postsynaptic currents (EPSCs) in the afferent fiber that translate into a phase-locked pattern of action potential activity. The present article summarizes our current understanding on the elementary properties of single IHC Ca2+ channels, and how these could have functional implications for certain, poorly understood, features of synaptic transmission at auditory hair cell ribbon synapses.

Mitochondrial Ca2+ uniporter (MCU)-dependent and MCU-independent Ca2+ channels coexist in the inner mitochondrial membrane

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca2+ uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca2+ channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca2+ and Na+ currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca2+ current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca2+ current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca2+ channels.

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca2+ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca2+, Mg2+, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca2+ sparks, Ca2+ blinks, and Ca2+ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca2+ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca2+ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca2+, as well as the time course of local Ca2+ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca2+ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca2+ spark initiation and subsequent Ca2+ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca2+ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca2+ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca2+ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca2+ sensitivities, Ca2+ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Single Ca2+ channels and exocytosis at sensory synapses

Hair cell synapses in the ear and photoreceptor synapses in the eye are the first synapses in the auditory and visual system. These specialized synapses transmit a large amount of sensory information in a fast and efficient manner. Moreover, both small and large signals with widely variable kinetics must be quickly encoded and reliably transmitted to allow an animal to rapidly monitor and react to its environment. Here we briefly review some aspects of these primary synapses, which are characterized by a synaptic ribbon in their active zones of transmitter release. We propose that these synapses are themselves highly specialized for the task at hand. Photoreceptor and bipolar cell ribbon synapses in the retina appear to have versatile properties that permit both tonic and phasic transmitter release. This allows them to transmit changes of both luminance and contrast within a visual field at different ambient light levels. By contrast, hair cell ribbon synapses are specialized for a highly synchronous form of multivesicular release that may be critical for phase locking to low-frequency sound-evoked signals at both low and high sound intensities. The microarchitecture of a hair cell synapse may be such that the opening of a single Ca(2+) channel evokes the simultaneous exocytosis of multiple synaptic vesicles. Thus, the differing demands of sensory encoding in the eye and ear generate diverse designs and capabilities for their ribbon synapses.

Modeling the Effect of Unitary Calcium Current on Neighboring Ryanodine Receptors during Calcium Induced Calcium Release

In resting cardiac cells, the open probability (Po) of single ryanodine receptors (RyRs) is very low and consequently little Ca2+ is released from the sarcoplasmic reticulum (SR). However, a stochastic RyR opening will cause diastolic local Ca2+ release from the SR that can activate neighboring closed RyRs. This inter-RyR Ca2+-induced Ca2+ release (CICR) may generate diastolic Ca2+ sparks. It is known that elevating SR Ca2+ load above normal levels dramatically increases spark frequency and increases the unitary RyR Ca2+ current. It is this current that acts on neighboring RyRs through CICR. We have developed a simple model based on experimental single-channel RyR Ca2+ sensitivity to understand how unitary RyR Ca2+ current may control CICR within a group of neighboring RyRs (a Ca2+ release unit, CRU). The model predicts how the current carried by an open RyR influences the activity of neighboring RyRs in a CRU. These predictions match published experimental single and clustered RyR channel results obtained in bilayer studies.

Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release

The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca2+ release from intracellular Ca2+ stores. How RyR's selectivity between the physiological cations K+, Mg2+, and Ca2+ affects single-channel Ca2+ current amplitude is examined using a recent model of RyR permeation. It is found that K+ provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca2+ release. Moreover, intra-pore competition between Ca2+ and Mg2+ defines single RyR Ca2+ current amplitude. Since both [Mg2+] and [Ca2+]SR can change during pathophysiological conditions, the RyR unitary Ca2+ current amplitude during Ca2+ release may change significantly due to this Ca2+/Mg2+ competition. Compared to the classic action of Mg2+ on RyR open probability, these Ca2+ current amplitude changes have as large or larger effects on overall RyR Ca2+ mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.

Modeling Ca2+ Induced Ca2+ Release Between Neighboring Ryanodine Receptors

In the heart, Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR). A small Ca2+ trigger activates a RyR, which then mediates a larger Ca2+ flux sufficient to activate neighboring RyRs. The inherent positive feedback of Ca2+ induced Ca2+ release (CICR) should cause Ca2+ release to continue until the SR is empty. This phenomenon does not occur in cells. In order to understand why, we have developed a simple physical model to describe CICR using simulations of discrete RyR channels on a two dimensional grid. RyR open probability (Po) is defined through a traditional two-site Hill function comprised of one cytosolic Ca2+ activation and one cytosolic Ca2+ inactivation sites per subunit. The diffusion equation for a steady state point source of Ca2+ current defines the Ca2+ concentration everywhere in the system. Single channel experimental Ca2+ dissociation constant values were used. Our Metropolis Monte Carlo simulations quantitatively reproduce Po's measured as a function of cytosolic Ca2+ for both single RyR as well as a two-RyR channel system reconstituted in bilayers. Our simulations suggest a mechanism for CICR termination requiring only a reduction in unitary Ca2+ current. Small changes in Ca2+ current produce large and sudden changes in overall activity in RyR arrays. Our simulations show reducing current may terminate release well before depletion of the SR at Ca2+ loads consistent with experiment. This current-termination occurs independently from any form of luminal regulation or coupled gating. Thus, we propose CICR termination may be due to a drop in single channel Ca2+ current as local intra-SR Ca2+ levels fall during release.

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function.