Electrostatic mechanisms underlie neomycin block of the cardiac ryanodine receptor channel (RyR2)

Neomycin is a large, positively charged, aminoglycoside antibiotic that has previously been shown to induce a voltage-dependent substate block in the cardiac isoform of the ryanodine receptor (RyR2). It was proposed that block involved an electrostatic interaction between neomycin and putative regions of negative charge in both the cytosolic and luminal mouths of the pore. In this study, we have attempted to screen charge by increasing potassium concentration in single-channel experiments. Neomycin block is apparent at both cytosolic and luminal faces of the channel in all K+ concentrations tested and alterations in K+ concentration have no effect on the amplitudes of the neomycin-induced substates. However, the kinetics of both cytosolic and luminal block are sensitive to changes in K+ concentration. In both cases increasing the K+ concentration leads to an increase in dissociation constant (KD). Underlying these changes are marked increases in rates of dissociation (k(off)), with little change in rates of association (k(on)). The increase in k(off) is more marked at the luminal face of the channel. Changes in K+ concentration also result in alterations in the voltage dependence of block. We have interpreted these data as supporting the proposal that neomycin block of RyR2 involves electrostatic interactions with the polycation forming a poorly fitting "plug" in the mouths of the conduction pathway. These observations emphasize the usefulness of neomycin as a probe for regions of charge in both the cytosolic and luminal mouths of the RyR2 pore.     

Ryanodine-induced structural alterations in the RyR channel suggested by neomycin block

In Mead and Williams, (Biophys. J. 82:1953-1963, 2002) we have reported that neomycin is a potent partial blocker of single purified sheep cardiac SR calcium release channels. Neomycin is unusual in that it is capable of blocking when applied to either the cytosolic or the luminal face of the channel. Block at either aspect of the channel is both concentration- and voltage-dependent, but exhibits different blocking parameters. In this study we have investigated the actions of neomycin on ion handling in the ryanodine-modified channel. Neomycin is more effective at the cytosolic face, having a Kb(0) value of 534.9 +/- 35.17 nM compared with a Kb(0) value of 971.5 +/- 66.62 nM for the luminal face. The voltage dependence also differs at the two sites. Values of zdelta for cytosolic and luminal neomycin are 1.09 +/- 0.04 and -0.57 +/- 0.03, respectively. The interaction of neomycin with the ryanodine-modified channel differs notably from that in the unmodified channel. Voltage-dependent relief of block is not observed after ryanodine modification, and the luminal blocking characteristics are altered. This suggests that ryanodine induces changes at the luminal mouth of the channel and may confer increased rigidity to the channel protein.     

Probing luminal negative charge in the type 3 ryanodine receptor

In this study, we have investigated block of potassium (K(+)) current by neomycin, a large polycation, from the luminal face of the type 3 ryanodine receptor (RyR3). Previous studies have shown that neomycin is an open channel blocker of RyR2 that interacts with negatively charged residues in the mouth of the conduction pathway to partially occlude it. In the current study, we have used neomycin as a probe to investigate proposed negatively charged regions in the luminal pore mouth of RyR3. Luminal neomycin induces concentration- and voltage-dependent partial block to a subconductance state in RyR3. Blocking parameters calculated in this study show that neomycin has a higher affinity for RyR3 than RyR2, but block may occur at the same site within the pore mouth. The change in affinity may be due to altered negative charge density at the site of interaction.     

The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site

In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.     

Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites.

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.     

Modulation of cardiac ryanodine receptor channels by alkaline earth cations

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.     

Ruthenium red modifies the cardiac and skeletal muscle Ca(2+) release channels (ryanodine receptors) by multiple mechanisms.

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca(2+) flux, [(3)H]ryanodine binding, and single channel measurements. In vesicle-Ca(2+) flux measurements, RR was more effective in inhibiting RyRs at 0.2 microM than 20 microM free Ca(2+). [(3)H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca(2+) regulatory sites of RyRs. In symmetric 0.25 M KCl with 10-20 microM cytosolic Ca(2+), cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 microM Ca(2+) + 2 mM ATP) and cardiac (200 microM Ca(2+)) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca(2+)-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes

The tetrameric ryanodine receptor calcium release channels (RyRs) are cation-selective channels that have pore architecture similar to that of K+ channels. We recently identified, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal DE motif that has a critical role in RyR ion permeation and selectivity. Here, we substituted three aspartate residues (D4938, D4945, D4953) with asparagine and four glutamate residues (E4942, E4948, E4952, E4955) with glutamine hypothesized to line the cytosolic vestibule of the skeletal muscle RyR (RyR1). Mutant single channel properties were determined using the planar lipid bilayer method. Two mutants (D4938N, D4945N) showed a reduced K+ ion conductance, with D4938N also exhibiting a reduced selectivity for Ca2+ compared to K+. The cytosolic location of D4938 and D4945 was confirmed using the polycation neomycin. Both D4938N and D4945N exhibited an attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D4899Q, E4900N) eliminated the block from the lumenal but not the cytosolic side. The results suggest that, in addition to negatively charged residues on the lumenal side, rings of four negative charges formed by D4938 and D4945 in the cytosolic vestibule determine RyR ion fluxes.     

Luminal Ca2+ regulation of single cardiac ryanodine receptors: insights provided by calsequestrin and its mutants

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.     

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Negative Charge at the Luminal Mouth of the Pore Alter Ion Handling and Gating in the Cardiac Ryanodine-Receptor

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K⁺ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.     

Spermine and arcaine block and permeate N-methyl-D-aspartate receptor channels.

Polyamines such as spermine are thought to be endogenous regulators of NMDA (N-methyl-D-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na+ concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from -60 to +60 mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of -120 to +60 mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.     

Two barium binding sites on a maxi K+ channel from human vas deferens epithelial cells.

Using the patch clamp technique, we have investigated the blockade of maxi-K+ channels present on vas deferens epithelial cells by extracellular Ba2+. With symmetrical 140 mM K+ solutions, Ba2+ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an "open channel blocking" scheme and was eliminated by reducing external K+ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K+ concentration. These data reveal that the vas deferens maxi-K+ channel has two Ba2+ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.     

Identification of Changes in the Functional Profile of the Cardiac Ryanodine Receptor Caused by the Coupled Gating Phenomenon

The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca²⁺ with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca²⁺ was used as a charge carrier, Ca²⁺-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca²⁺. Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca²⁺ current amplitude-luminal Ca²⁺ relationship displayed a simple saturation and the channel selectivity for Ba²⁺ and Ca²⁺ ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca²⁺ fluxes.     

Modification of cardiac RYR2 gating by a peptide from the central domain of the RYR2

The effect of a domain peptide DP_CPVTc from the central region of the RYR2 on ryanodine receptors from rat heart has been examined in planar lipid bilayers. At a zero holding potential and at 8 mmol L^?1 luminal Ca²⁺ concentration, DP_CPVTc induced concentrationdependent activation of the ryanodine receptor that led up to 20-fold increase of P_O at saturating DP_CPVTc concentrations. DP_CPVTc prolonged RyR2 openings and increased RyR2 opening frequency. At all peptide concentrations the channels displayed large variability in open probability, open time and frequency of openings. With increasing peptide concentration, the fraction of high open probability records increased together with their open time. The closed times of neither low- nor high-open probability records depended on peptide concentration. The concentration dependence of all gating parameters had EC₅₀ of 20 μmol L^?1 and a Hill slope of 2. Comparison of the effects of DP_CPVTc with the effects of ATP and cytosolic Ca²⁺ suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DP_CPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

Ryanodine Receptor Luminal Ca Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca²⁺ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca²⁺ channel and the intra-SR Ca²⁺ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca²⁺ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca²⁺ dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca²⁺) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca²⁺ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca²⁺ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca²⁺ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca²⁺ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca²⁺ buffer.     

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.