Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca2+ channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR α1S or β1a subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the ∼60-kD streptavidin molecule had access to the β1a N and C termini and to the α1S N terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the α1S C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the β1a N or C terminus, or to the α1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α1S II–III loop abolished such contractions, without affecting agonist-induced Ca2+ release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the α1S II–III loop are necessary for EC coupling in skeletal muscle

Potentiated L-type Ca2+ Channels Rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type α1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-α1C) or repeat III (E3K-α1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-α1C and E3K-α1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (−)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-α1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-α1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca2+ entry (ECCE) designates the entry of extracellular Ca2+ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca2+ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn2+. Here, we have examined the effects of these agents on the L-type Ca2+ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La3+, and Gd3+) and dantrolene all inhibited the L-type Ca2+ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca2+ channel displays permeability to Mn2+ in the absence of external Ca2+ and produces a Ca2+ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca2+ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca2+; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca2+-free solution and were unaffected by Ca2+ removal; and (3) after block of SR Ca2+ release by 200 μM ryanodine, normal myotubes still displayed a large Ca2+ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca2+ entry attributed to ECCE

A malignant hyperthermia–inducing mutation in RYR1 (R163C): consequent alterations in the functional properties of DHPR channels

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation–contraction coupling (voltage-gated Ca2+ release from the SR) and increased amplitude of L-type Ca2+ current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca2+ release from the SR, membrane-bound charge movement, and/or L-type Ca2+ current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca2+ release from the SR was substantially reduced and shifted (∼10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca2+ release. The current–voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials (∼7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist ±Bay K 8644 (10 µM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to −50, −10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes

Effects of inserting fluorescent proteins into the α1S II–III loop: insights into excitation–contraction coupling

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca2+ release via RYR1, and retrograde coupling is manifested by increased L-type Ca2+ current via DHPR. A critical domain (residues 720–765) of the DHPR α1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the α1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca2+ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the α1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the α1S II–III loop. Significantly, our results indicate that the conserved portion of the α1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1

A malignant hyperthermia–inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR

Bidirectional signaling between the sarcolemmal L-type Ca2+ channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca2+ release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation–contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K+ depolarization. The new findings are: (a) For all three genotypes, Ca2+ transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca2+ or the addition of Ca2+ entry blockers (nifedipine, SKF96365, and Ni2+) enhanced the rate of decay in all genotypes. (d) When Ca2+ entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K+ depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor

Scientific Opportunities for Monitoring at Environmental Remediation Sites (SOMERS): Integrated Systems-Based Approaches to Monitoring

Through an inter-disciplinary effort, DOE is addressing a need to advance monitoring approaches from sole reliance on cost- and labor-intensive point-source monitoring to integrated systems-based approaches such as flux-based approaches and the use of early indicator parameters. Key objectives include identifying current scientific, technical and implementation opportunities and challenges, prioritizing science and technology strategies to meet current needs within the DOE complex for the most challenging environments, and developing an integrated and risk-informed monitoring framework

The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation

Muscular dysgenesis ( mdg) is a mutation in mice which causes the failure of excitation-contraction coupling in skeletal muscle. Although the sarcolemma, the sarcoplasmic reticulum, and the contractile apparatus all maintain nearly normal function, sarcolemmal depolarization fails to cause calcium release from the sarcoplasmic reticulum. Recently, the primary genetic defect in this mutation was shown to be located in the structural gene for the dihydro-pyridine receptor. We have examined the developmental expression from Fetal Day 15 onward, in normal and mutant muscle, of several unidentified genes as well as genes which are known markers of muscle differentiation. We find that the majority of mRNA sequences are found at similar concentrations in normal and dysgenic muscles at birth. Many differentiation-related genes also are expressed at normal levels early during myogenesis in mutant mice. However, as late fetal development progresses in dysgenic muscle, the mRNA concentrations for these genes fail to undergo the rapid rise which is characteristic of normal muscle. Several additional, unidentified genes, which normally would be down-regulated during development, remain expressed at a high level in dysgenic muscle. Thus, the primary absence of a functional dihydropyridine receptor appears to prevent the changes in gene expression which are necessary for maturation of skeletal muscle

mRNA for Cardiac Calcium Channel Is Expressed during Development of Skeletal Muscle

During the early development of skeletal muscle, cardiac isotypes of several contractile proteins are known to be transiently expressed. We report here that skeletal muscle developing in vivo, as well as primary cultures derived from skeletal muscle, express mRNA encoding the cardiac dihydropyridine-sensitive calcium channel. The mRNA is detectable at high concentration at the earliest stage tested in vivo and diminishes rapidly in concentration as myofibers mature. The concentration of the cardiac calcium channel mRNA also diminishes during the in vivo development of skeletal muscle in a genetically paralyzed mouse ( mdg), indicating that muscle contractile activity is not necessary for the down-regulation. In contrast, mRNA for the skeletal muscle-specific calcium channel accumulates gradually in developing skeletal muscle. A similar temporal pattern of expression is also seen in primary cultures of skeletal myotubes. These results raise the question of whether the cardiac calcium channel may be functionally important during the early development of skeletal myofibers