Manfred Grabner

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 Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca2+ release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca2+ 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 Ca2+currents. Introducing a short skeletal segment (α1Sresidues 720–765) into the cardiac II-III loop (replacing α1C residues 851–896) of GFP-SkLC restored both EC coupling and Ca2+ 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 Ca2+ current.

Transfer of 1,4-Dihydropyridine Sensitivity from L-Type to Class A (BI) Calcium Channels

L-type Ca2+ channels are characterized by their unique sensitivity to organic Ca2+ channel modulators like the 1,4-dihydropyridines (DHPs). To identify molecular motifs mediating DHP sensitivity, we transferred this sensitivity from L-type Ca2+ channels to the DHP-insensitive class A brain Ca2+ channel, BI-2. Expression of chimeras revealed minimum sequence stretches conferring DHP sensitivity including segments IIIS5, IIIS6, and the connecting linker, as well as the IVS5-IVS6 linker plus segment IVS6. DHP agonist and antagonist effects are determined by different regions within the repeat IV motif. Sequence regions responsible for DHP sensitivity comprise only 9.4% of the overall primary structure of a DHP-sensitive alpha 1A/alpha 1S construct. This chimera fully exhibits the DHP sensitivity of channels formed by L-type alpha 1 subunits. In addition, it displays the electrophysiological properties of alpha 1A, as well as its sensitivity toward the peptide toxins omega-agatoxin IVA and omega-conotoxin MVIIC.

Endogenous calcium channels in human embryonic kidney (HEK293) cells.

1 We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr‐HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr‐HEK were characterized and compared with the properties of a heterologously expressed chimeric L‐type calcium channel construct. 2 ISr‐HEK activated on depolarization to voltages positive of −40 mV. It had transient current kinetics with a time to peak of 16 ± 1.4 ms (n = 7) and an inactivation times constant of 52 ± 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was −19.0 ± 1.5 mV (n = 7) and the voltage for half maximal steady‐state inactivation was −39.7 ± 2.3 mV (n = 7). 3 Block of ISr‐HEK by the dihydropyridine isradipine was not stereoselective; 1 μm (+) and (−)−isradipine inhibited the current by 30 ± 4% (n = 3) and 29 ± 2% (n = 4) respectively. (+)‐Isradipine and (−)−isradipine (10 μm) inhibited ISr‐HEK by 89 ± 4% (n = 5) and 88 ± 8% (n = 3) respectively. The 7‐bromo substituted (±)‐isradipine (VO2605, 10 μm) which is almost inactive on L‐type calcium channels also inhibited ISr‐HEK (83 ± 9%, n = 3) as was observed for 10 μm (−)−nimodipine (78 ± 6%, n = 5). Interestingly, 10 μm (±)‐Bay K 8644 (n = 5) had no effect on the current. ISr‐HEK was only slightly inhibited by the cone snail toxins ω‐CTx GVIA (1 μm, inhibition by 17 ± 3%, n = 4) and ω‐CTx MVIIC (1 μm, inhibition by 20 ± 3%, n = 4). The funnel web spider toxin ω‐Aga IVA (200 nM) inhibited ISr‐HEK by 19 ± 2%, n = 4). 4 In cells expressing ISr‐HEK, maximum inward current densities of 0.24 ± 0.03 pA/pF and 0.39 ± 0.7 pA/pF (at a test potential of −10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 ± 0.18 pA/pF or 1.11 ± 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum‐free medium. 5 Co‐expression of a chimeric L‐type calcium channel construct revealed that ISr‐HEK and L‐type calcium channel currents could be distinguished by their different voltage‐dependencies and current kinetics. The current density after heterologous expression of the L‐type α1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 ± 0.45 pA/pF) than that of ISr‐HEK under the same conditions.

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel

The tritiated arylazido phenylalkylamine (‐)‐5‐[(3‐azidophenethyl)[N‐methyl‐3H]methylamino]‐2‐(3,4,5‐tri‐methoxyphenyl)‐2‐isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane‐bound and purified calcium channel from guinea‐pig skeletal muscle trans‐verse‐tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a K d of 2.0 ± 0.5 nM and a B max of 17.0 ± 0.9 protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse‐tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver‐stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high‐affinity drug receptor carrying subunit of the skeletal muscle transverse‐tubule L‐type calcium channel.

Transfer of High Sensitivity for Benzothiazepines from L-type to Class A (BI) Calcium Channels

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (α1S or α1C) to a weakly sensitive, nonstereoselective class A (α1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type α1 into the α1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (−)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (−)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α1S II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α1S 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 (Ca2+ 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 α1S) has no similarity to α1S in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca2+ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α1S 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 α1S loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α1S-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.

Two Amino Acid Residues in the IIIS5 Segment of L-Type Calcium Channels Differentially Contribute to 1,4-Dihydropyridine Sensitivity

The transmembrane segment IIIS5 of the L-type calcium channel α1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel α1 subunits with α2δ and β1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to α1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel α1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative α-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β1-null Zebrafish Relaxed Is an Exclusive Function of the β1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β1a subunit. Lack of β1a results in (i) reduced membrane expression of the pore forming DHPR α1S subunit, (ii) elimination of α1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β1a from rather general functions of β isoforms. Zebrafish and mammalian β1a subunits quantitatively restored α1S triad targeting and charge movement as well as intracellular Ca2+ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β2a as the phylogenetically closest, and the ancestral housefly βM as the most distant isoform to β1a also completely recovered α1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca2+ transients and very limited tetrad formation compared with β1a. Consequently, larval motility was either only partially restored (β2a-injected larvae) or not restored at all (βM). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β1a isoform. Consequently, we postulate a model that presents β1a as an allosteric modifier of α1S conformation enabling skeletal muscle-type EC coupling.

Molecular Basis of Drug Interaction with L-Type Ca2+ Channels

Different types of voltage-gated Ca2+ channels exist in the plasma membrane of electrically excitable cells. By controlling depolarization-induced Ca2+ entry into cells they serve important physiological functions, such as excitation-contraction coupling, neurotransmitter and hormone secretion, and neuronal plasticity. Their function is fine-tuned by a variety of modulators, such as enzymes and G-proteins. Block of so-called L-type Ca2+ channels by drugs is exploited as a therapeutic principle to treat cardiovascular disorders, such as hypertension. More recently, block of so-called non-L-type Ca2+ channels was found to exert therapeutic effects in the treatment of severe pain and ischemic stroke. As the subunits of different Ca2+ channel types have been cloned, the modulatory sites for enzymes, G-proteins, and drugs can now be determined using molecular engineering and heterologous expression. Here we summarize recent work that has allowed us to determine the sites of action of L-type Ca2+ channel modulators. Together with previous biochemical, electrophysiological, and drug binding data these results provide exciting insight into the molecular pharmacology of this voltage-gated Ca2+ channel family.

Calcium channels: The β‐subunit increases the affinity of dihydropyridine and Ca2+ binding sites of the α1‐subunit

A Ca2+ channel α1‐subunit derived from rabbit heart was transiently expressed in COS‐7 cells. The dihydropyridine (+)‐isradipine had low affinity (K i = 34.3 nM) for the α1‐subunit in the absence of the β‐subunit due to rapid dissociation (k −1 = 0.11 min−1). Co‐expression of the β‐subunit resulted in a ⪢ 35‐fold increase in (+)‐isradipine binding affinity (K i = 0.9 nM) due to decreased dissociation (k −1 of 0.007 min−1). Higher DHP binding affinity was associated with an increase of the apparent affinity of Ca2+ ions for the channel. Our data suggest that the β‐subunit affects the coordination of Ca2+ ions with sites that are coupled to the dihydropyridine binding domain and by this mechanism increases the affinity for these ligands.