Biomedical subjects

K G Beam

Publications and source records attributed to K G Beam.

At least 19 recordsLinked to original sources

PubMed2001

Excitation-contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720-L764 of the alpha 1S II-III loop.

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1S) subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation-contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681-L690 and residues L720-Q765, respectively), claiming for each a key function in DHPR-RyR1 communication. To address whether residues 720-765 of the II-III loop are sufficient to enable skeletal-type (Ca(2+) entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to alpha(1S)) has no similarity to alpha(1S) in the regions R681-L690 and L720-Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous alpha(1S) subunits) was unable to restore EC coupling and displayed strongly reduced Ca(2+) current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit alpha(1S) residues L720-L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724-T755) completely restored bidirectional coupling, indicating its dependence on alpha(1S) loop residues 720-764 but its independence from other regions of the loop. Thus, 45 alpha(1S)-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein-protein interaction required for bidirectional coupling.

Amino Acid Sequence↗

PubMed2001

Structure and targeting of RyR1: implications from fusion of green fluorescent protein at the amino-terminal.

In skeletal muscle, an anterograde signal from the dihydropyridine receptor (DHPR) to the ryanodine receptor (RyR1) is required for excitation-contraction (EC) coupling and a retrograde signal from RyR1 to the DHPR regulates the magnitude of the calcium current carried by the DHPR. As a tool for studying biosynthesis and targeting, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the amino terminal of RyR1 and expressed it in dyspedic myotubes. The GFP-RyR1 was present in a restricted domain near the nucleus injected with cDNA and was fully functional, which places constraints on the location of the amino terminal in the folded structure of RyR1.

Animals↗

PubMed2001

The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins.

FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.

Amino Acid Sequence↗

PubMed2001

Potentiation of the cardiac L-type Ca(2+) channel (alpha(1C)) by dihydropyridine agonist and strong depolarization occur via distinct mechanisms.

A defining property of L-type Ca(2+) channels is their potentiation by both 1,4-dihydropyridine agonists and strong depolarization. In contrast, non-L-type channels are potentiated by neither agonist nor depolarization, suggesting that these two processes may by linked. In this study, we have tested whether the mechanisms of agonist- and depolarization-induced potentiation in the cardiac L-type channel (alpha(1C)) are linked. We found that the mutant L-type channel GFP-alpha(1C)(TQ-->YM), bearing the mutations T1066Y and Q1070M, was able to undergo depolarization-induced potentiation but not potentiation by agonist. Conversely, the chimeric channel GFP-CACC was potentiated by agonist but not by strong depolarization. These data indicate that the mechanisms of agonist- and depolarization-induced potentiation of alpha(1C) are distinct. Since neither GFP-CACC nor GFP-CCAA was potentiated significantly by depolarization, no single repeat of alpha(1C) appears to be responsible for depolarization-induced potentiation. Surprisingly, GFP-CACC displayed a low estimated open probability similar to that of the alpha(1C), but could not support depolarization-induced potentiation, demonstrating that a relatively low open probability alone is not sufficient for depolarization-induced potentiation to occur. Thus, depolarization-induced potentiation may be a global channel property requiring participation from all four homologous repeats.

Action Potentials↗

PubMed2000

Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop.

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

Amino Acid Sequence↗

PubMed2000

A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor.

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

Animals↗

PubMed2000

Low serum promotes maturation of excitation-contraction coupling in myotubes.

Patch-clamping and the simultaneous fluorescence measurement of cytoplasmic Ca2+ ([Ca2+]i) were used to analyze the effect of serum on the functional features of excitation-contraction (E-C) coupling in mouse skeletal myotubes. In high-serum-treated (10%) myotubes, depolarization elicited Ca2+ release which continued for tens of milliseconds following the end of the pulse, after which [Ca2+]i decayed slowly. In low-serum-treated (0.5%) myotubes, the Ca2+ transient caused by depolarization had an increased rate of rise and peak amplitude, and [Ca2+]i began to decay rapidly upon repolarization. When a depolarizing pulse (0.5-1.0 s) was applied to low-serum-treated myotubes during a Ca2+ transient induced by 5-10 mM caffeine, repolarization usually caused the caffeine transient to terminate rapidly (RISC; repolarization-induced stop of caffeine-induced Ca2+ release). The RISC was less prominent in high-serum-treated myotubes. These results suggest that low serum promotes the maturation of myotubes so that Ca(2+)-release and Ca(2+)-removal activities are accelerated. Additionally, the essential features of the communication between the voltage sensor and the Ca(2+)-release channel are shared by myotubes and adult muscle fibers.

Age Factors↗

PubMed2000

Calcium channelopathies.

Calcium is an important intracellular signaling molecule, and altered calcium channel function can cause widespread cellular changes. Genetic mutations in calcium channels that cause what appear to be trivial alterations of calcium currents in vitro can result in serious diseases in muscles and the nervous system. This article reviews calcium channelopathies in humans and mice.

Animals↗

PubMed1999

The absence of resurgent sodium current in mouse spinal neurons.

The Scn8a gene encodes a neuronal, voltage-gated sodium channel, which is highly expressed in both cerebellar Purkinje neurons and spinal motoneurons [D.L. Burgess, D.C. Kohrman, J. Galt, N.W. Plummer, J.M. Jones, B. Spear, M.H. Meisler, Mutation of a new sodium channel gene, Scn8a, in the mouse mutant 'motor endplate disease', Nature Genetics 10 (1995) 461-465; K.L. Schaller, D.M. Krzemien, P.J. Yarowsky, B.K. Krueger, J.H. Caldwell, A novel, abundant sodium channel expressed in neurons and glia, J. Neurosci. 15 (1995) 3231-3242]. Sodium channels in Purkinje cells produce an unusual, "resurgent" current when the cells are repolarized to intermediate potentials (-60 to -20 mV) following a strong depolarization that completely inactivates transient sodium current [I.M. Raman, L.K. Sprunger, M.H. Meisler, B.P. Bean, Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice, Neuron 19 (1997) 881-891; I.M. Raman, B.P. Bean, Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons, J. Neurosci. 17 (1997) 4517-4526]. Here, we have examined whether large spinal neurons (predominantly motoneurons), isolated from P6-P8 mice and cultured overnight, produce sodium currents resembling those either of Purkinje cells or of Xenopus oocytes after heterologous expression of Scn8a. We found that P10-P14 Purkinje cells exhibited resurgent current (ranging from -3.6 to -15.4 pA/pF in 16 cells at -40 mV), but cultured spinal neurons had little or no such current (<0.5 pA/pF in 13 of 16 cells; -1.2 to -2.3 pA/pF in three of 16 cells). Furthermore, unlike Scn8a channels heterologously expressed in Xenopus oocytes [M.R. Smith, R.D. Smith, N.W. Plummer, M.H. Meisler, A.L. Goldin, Functional analysis of the mouse Scn8a sodium channel. J. Neurosci. 18 (1998) 6093-6102], there was not a prominent component of persistent sodium current in either Purkinje neurons or large spinal neurons. Based on analysis of cells from mice with a Scn8a null mutation, Scn8a channels appear to contribute significantly to total sodium current in both in P10-P14 Purkinje cells (approximately 40%; [21]) and cultured P7-P8 spinal motoneurons (approximately 70% [K.D. García, L.K. Sprunger, M.H. Meisler, K.G. Beam, The sodium channel Scn8a is the major contributor to the postnatal developmental increase of sodium current density in spinal motoneurons, J. Neurosci. 18 (1998) 5234-5239]). Thus, the presence or absence of resurgent current, and of persistent sodium current, appears to depend on cellular factors other than the mere presence of the Scn8a transcript.

Action Potentials↗

PubMed1999

The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.

Amino Acid Sequence↗

PubMed1999

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.

Algorithms↗

PubMed1998

Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling.

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2+ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca2+ through the channel is not required for skeletal muscle-type EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser687 of the skeletal II-III loop has been reported to be rapidly phosphorylated in vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alanine-substituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725-742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725-742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711-765). However, residues 725-742 appeared to be critical because skeletal-type coupling was not produced either by a chimera with skeletal residues 711-732 or by one with skeletal residues 734-765.

Amino Acid Sequence↗

PubMed1998

The sodium channel Scn8a is the major contributor to the postnatal developmental increase of sodium current density in spinal motoneurons.

Sodium currents were recorded from motoneurons that were isolated from mice at postnatal days 0-8 (P0-P8) and maintained in culture for 12-24 hr. Motoneurons from normal mice exhibited a more than threefold increase in peak sodium current density from P0 to P8. For mice lacking a functional Scn8a sodium channel gene, motoneuronal sodium current density was comparable at P0 to that of normal mice but failed to increase from P0 to P8. The absence of Scn8a sodium channels is associated with the phenotype "motor end plate disease," which is characterized by a progressive neuromuscular failure and is fatal by 3-4 postnatal weeks. Thus, it appears that the development and function of mature motoneurons depends on the postnatal induction of Scn8a expression.

Animals↗

PubMed1998

Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leaner.

Mutations of the alpha1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes of tottering and leaner. The leaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Here, we have used whole-cell recordings to determine whether the leaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of alpha1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nM omega-agatoxin-IVA. In Purkinje cells from homozygous leaner mice, this omega-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the omega-agatoxin-IVA-sensitive current in homozygous leaner cells had properties indistinguishable from that of normal Purkinje neurons. Additionally, the omega-agatoxin-IVA-insensitive current was unaffected in homozygous leaner mice. Thus, the leaner mutation selectively reduces P-type currents in Purkinje cells, and the alpha1A subunit and P-type current appear to be essential for normal cerebellar function.

Animals↗

PubMed1998

Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels.

Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as L-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature 325, 717-720). In addition to "receiving" the EC coupling signal from the DHPR, RyR-1 also "transmits" a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72-76). A similar kind of retrograde signaling (from RyRs to L-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719-722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635-2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+ channel function, whereas a chimera containing adjacent RyR-1 residues (2, 659-3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Animals↗

PubMed1998

Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes.

Expression of cardiac L-type Ca2+ channels in dysgenic myotubes results in large Ca2+ currents and electrically evoked contractions resulting from Ca2+-entry dependent release of Ca2+ from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca2+ channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca2+ currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca2+ channels in close apposition to sarcoplasmic reticulum Ca2+ release channels. We tagged the N termini of different alpha1 subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged alpha1 subunit exhibited Ca2+ channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-alpha1S and GFP-alpha1C in dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-alpha1A and GFP-alpha1B failed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-alpha1S and GFP-alpha1C were present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-alpha1A and GFP-alpha1B were not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.

Animals↗

PubMed1997

Functional nonequality of the cardiac and skeletal ryanodine receptors.

Dihydropyridine receptors (DHPRs), which are voltage-gated Ca2+ channels, and ryanodine receptors (RyRs), which are intracellular Ca2+ release channels, are expressed in diverse cell types, including skeletal and cardiac muscle. In skeletal muscle, there appears to be reciprocal signaling between the skeletal isoforms of both the DHPR and the RyR (RyR-1), such that Ca2+ release activity of RyR-1 is controlled by the DHPR and Ca2+ channel activity of the DHPR is controlled by RyR-1. Dyspedic skeletal muscle cells, which do not express RyR-1, lack excitation-contraction coupling and have an approximately 30-fold reduction in L-type Ca2+ current density. Here we have examined the ability of the predominant cardiac and brain RyR isoform, RyR-2, to substitute for RyR-1 in interacting with the skeletal DHPR. When RyR-2 is expressed in dyspedic muscle cells, it gives rise to spontaneous intracellular Ca2+ oscillations and supports Ca2+ entry-induced Ca2+ release. However, unlike RyR-1, the expressed RyR-2 does not increase the Ca2+ channel activity of the DHPR, nor is the gating of RyR-2 controlled by the skeletal DHPR. Thus, the ability to participate in skeletal-type reciprocal signaling appears to be a unique feature of RyR-1.

Animals↗

PubMed1997

The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel conductance.

The alpha1-subunits of the skeletal and cardiac L-type calcium channels (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high conservation of the P-regions, native skeletal L-channels exhibit a unitary conductance that is only about half that of native cardiac L-channels. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeric L-channel alpha1-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-IS6 linker) is a critical determinant of the difference in skeletal and cardiac unitary conductance. The unitary conductances attributable to the wild-type skeletal (CAC6; approximately 14 pS) and cardiac (CARD1; approximately 25 pS) alpha1-subunits expressed in dysgenic myotubes are identical to those observed in native tissues. Chimeric alpha1-subunits containing skeletal sequence for the first internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the IS5-IS6 linker (SkC49) each exhibit a low, skeletal-like unitary conductance (< or = 17 pS). Constructs in which the IS5-IS6 linker is of cardiac origin (CARD1 and CSk9) display cardiac-like conductance (approximately 25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SkC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-IS6 linker strongly influences the single channel conductance of L-channels in a manner that is independent from the rate of channel activation.

Amino Acid Sequence↗