Insect calcium channels

Abstract

The complete ammo acid sequence of an invertebrate calcium channel ~,-subunit from housefly (Musca domestrca) larvae (designated Mdla,) has been deduced by cDNA cloning and sequence analysis. Mdb, shares higher percent sequence identity with 1,4-dihydropy~dine (DHP)-sensitive L-type than with DHP-insensitive calcium channels. As shown by whole mount in situ hybridization and immunostaining Mdla, is predominantly expressed in the larval body wall musculature.Key words: Calcium channel; cDNA cloning; Hybridization (in situ); Body wall muscle; Phylogenetic tree I. Int~uctiouIntracellular free Ca'+ acts as a second messenger for a variety of physiological processes. In electrically excitable cells Ca2' entry is mediated by different types (L-, N-, P-and T-type) of voltage-activated calcium channels, which are distinguished in vertebrates by biophysical and pharmacological criteria (for a review see [I]). These channels exist as hetero-oligomeric complexes of several subunits (a,, a,-&, /I and y-subunits), which have been cloned and sequenced from various tissues 12-53. a,-Subunits form the channel pore and are structurally homologous to the channel-forming subunits of voltage-activated sodium and potassium channels. Phylogenetic analyses of a,-subunits from different types of vertebrate calcium channels suggest that they evolved from a common ancestor gene, which diverged into at least two main subfamilies [ 11. One subfamily, classified as L-type channels according to electrophysiological criteria [6], is sensitive to calcium channel drugs, like 1,4-dihydropyridines (DHP), phenylalkylamines (PAA) and benzothiazepines (reviewed in [7]). All other calcium channel *Corresponding author. Fax: (43) (512) 588 627.Abbreviations: DHP, dihydropyridine; DIG, digoxigenin-1 l-dUTP; Mdla,, Musca domestica larvae a,-subunit; PAA, phenylalkylamines; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate. types are not DHP-sensitive but can be distinguished by toxins (for review see [8]).Much less is known about the structure and pharmacological sensitivity of voltage-gated calcium channels in insects, which are separated from vertebrates by about 600 million years in evolution [9]. In arthropods different voltagegated calcium channels play a key role for muscle contraction: presynaptic channels control the release of excitatory transmitter (mainly glutamate) [lO,l l] at the neuromuscular junction, whereas calcium channels in the plasma membrane of muscle cells mediate the influx of calcium ions for contraction and participate in the propagation of the action potential [12]. The structural characterization of invertebrate calcium channels is rudimentary [13][14][15] and none of their subunits have yet been cloned.Here we report for the first time the cloning of an invertebrate calcium channel a,-subunit from Musea domestica larvae (designated Mdla,) that is preferentially expressed in body wall muscle. The evolutionary relationship of Mdla, compared to the so far cloned vertebrate calcium channels has been studied by...

Nine L-type Amino Acid Residues Confer Full 1,4-Dihydropyridine Sensitivity to the Neuronal Calcium Channel α1ASubunit

“…The importance of an additional L-type methionine residue (Met 1188 ; ␣ 1C-a numbering) to fully support DHP sensitivity represents a new finding presented in this study. The role of Met 1188 was uncovered by the differences in DHP agonist and antagonist sensitivity of the chimeric calcium channel ␣ 1 subunit AL12m, that was constructed by merging sequence stretches from the DHP-insensitive ␣ 1A subunit (22) and the ␣ 1M subunit, which had been cloned from M. domestica body wall muscle (24). AL12m, in addition to having provided an opportunity to identify the importance of Met 1188 for DHP interaction, enabled us to characterize for the first time the DHP sensitivity of an ancestral L-type ␣ 1 subunit (Fig.…”

“…Residue in IIIS6 for DHP Sensitivity-Recently, we have cloned the ancestral L-type ␣ 1M subunit from housefly (M. domestica) body wall muscle (24). Similar to L-type ␣ 1 subunits derived from vertebrate skeletal muscle (6,31), functional expression of ␣ 1M in Xenopus oocytes failed (24).…”

“…Similar to L-type ␣ 1 subunits derived from vertebrate skeletal muscle (6,31), functional expression of ␣ 1M in Xenopus oocytes failed (24). We therefore attempted to investigate the DHP sensitivity of ␣ 1M by means of a chimeric calcium channel ␣ 1 subunit, termed AL12m ( Fig.…”

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

“…The EcoRI-BalI fragment of the rabbit skeletal muscle DHPR ␣ 1S subunit (Sk) (nucleotides 1007-1973) was coligated with the BalI-NdeI fragment (nucleotides 1982-2296) from the II-III loop (L) from the body-wall muscle DHPR ␣ 1 subunit (M) of M. domestica (17) into plasmid pSP72 (Promega) by using the internal NdeI site (plasmid nucleotide 2379) and the EcoRI site of the polylinker. The NdeI͞EcoRI restriction sites of pSP72 also were used to coligate two cDNA fragments, the NdeI*-XhoI fragment that was PCR-generated from the clone GFP-SkLC, GFP-␣ 1S with the cardiac (␣ 1C, C) II-III loop (nucleotides C2716-Sk2654) (9), plus the XhoI-BglII fragment of Sk (nucleotides 2654-4488).…”

“…To accomplish this, we created the chimera GFP-SkLM (Fig. 1 A), in which the II-III loop of ␣ 1S was replaced by the highly divergent II-III loop of a DHPR cloned from the housefly (M. domestica) (17). Although we have not been able to express the Musca ␣ 1 subunit functionally in various heterologous systems (Xenopus oocytes, tsA-201 cells, or dysgenic myotubes), we did find that constructs containing parts of the Musca DHPR sequence were valuable for fine mapping of the DHP-binding domain (29).…”