Properties of the α1-β Anchoring Site in Voltage-dependent Ca2+ Channels

Abstract

In voltage-dependent Ca2+ channels, the beta subunit interacts with the alpha 1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized 35S-labeled beta subunits specifically bind to a glutathione S-transferase (GST) fusion protein containing the alpha 1A interaction domain (AIDA, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of 35S-labeled beta 1b subunit to the AIDA GST fusion protein occurs with a fast rate constant at 4 degrees C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AIDA peptide. The alpha 1-beta binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca2+ concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca2+ channels was also followed by saturation analyses. The data obtained show that the AIDA GST fusion protein binds to a single site on the beta 1b with an apparent Kd of 5 nM. The affinities of AIDA GST fusion protein for various beta subunits was measured and demonstrate that beta subunits associate with different affinities to each alpha 1 interaction domain. The rank order of AIDA affinity for each beta subunit is as follows: beta 4 > beta 2a > beta 1b >> beta 3. The binding of the beta subunit to alpha 1 subunit can be inhibited in vitro by the AIDA synthetic peptide with an apparent Ki of 285 nM. This interaction can also be prevented in heterologous Ca2+ channels by the injection of the AIDA GST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and beta subunit is responsible for anchoring the beta subunit to the alpha 1 subunit and thus allowing the beta subunit to modify Ca2+ channel activity.

Identification of critical amino acids involved in α1‐β interaction in voltage‐dependent Ca2+ channels

“…lb). At this saturating concentration (100 nM), as previously determined [21], the wild-type AIDg-Sepharose beads bound approximately 57% of the total in vitro translated [35S]fllb protein. This fraction was comparable to the amount of [35S1B~b (41.4 _ 4.5%) that could be immunoprecipitated by VD2~ a monoclonal antibody that recognizes a conserved sequence in fl subunits (data not shown).…”

“…The high-affinity Kd values were 5.6 nM (~IbP221R), 7.2 nM (J~lb $228A), 5.8 nM (fllb $238A) and 7.9 nM (fllb G241R) which in all cases were not significantly different from the 5.8 nM K d value of the wild-type Btb subunit [21]. It was previously shown on the basis of coexpression experiments that AID or BID point mutations which significantly modify the ~-B interaction in vitro also prevented their native functional association in Xenopus oocytes [4][5].…”

“…To examine the specific interaction between the cq and fl subunits, we have developed an in vitro binding assay [21]. In this affinity assay, the AIDA is expressed as a 50 amino acid GST fusion protein and coupled at various concentrations to glutathione-Sepharose beads to form a ligand defined herein as AIDA-Sepharose beads.…”

“…Purification of the Glutathione S-Transferase (GST) fusion proteins and binding of these fusion proteins to the wild-type or mutant flt b subunits were performed as described previously [21]. Xenopus laevis oocyte preparation and maintenance, in vitro transcription, and cRNA injection were performed according to protocols described elsewhere [24]. Briefly, 50 nl of various transcribed cRNAs were injected into each oocyte at the following concentrations (flg//ll): cqA (0.4) and wild-type or mutant fllb (0.2).…”

“…In an alternative mechanism, the role of fl subunits may be to increase the number of functional col subunits at the plasma membrane, although changes in kinetics and voltage-dependent parameters and some slight changes in opening probability as reported [18] would be the result of somewhat more subtle conformational changes. The present identification of critical residues in BID and the recent development of a biochemical assay to measure the affinity between ~1 and fl subunits [21], provided a unique alternative to examine further either one of these hypotheses. Herein, we have investigated the role of several as yet uncharacterized AID and BID amino acids in the anchoring of the fl subunits to ~1 channels.…”