Barry D. Johnson

Primary Structure and Function of an A Kinase Anchoring Protein Associated with Calcium Channels

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

Molecular Determinants of High Affinity Phenylalkylamine Block of L-type Calcium Channels

The high affinity phenylalkylamine(-)D888 blocks ion currents through L-type Ca2+ channels containing the α1C subunit with an apparent Kdof 50 nM, but N-type Ca2+ channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher Kd value of 5 μM. L-type Ca2+ channels containing α1C subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV (IVS6) were 6-12 times less sensitive to block by(-)D888 than control α1C. Ca2+ channels containing paired combinations of these mutations were even less sensitive to block by(-)D888 than the single mutants, and channels containing all three mutations were >100 times less sensitive to(-)D888 block, similar to N-type Ca2+ channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity, suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues are aligned on the same face of the putative IVS6 α-helix, we propose that they contribute to a receptor site in the pore that confers a high affinity block of L-type channels by(-)D888.

Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Recent studies of the phenylalkylamine binding site in the α1C subunit of l-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to l-type channels. We have extended this analysis of the phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the β1b and α2δ auxiliary subunits. Assay for block of Ba2+ current by (−)-D888 at −60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between l-type and non-l-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one l-type-specific amino acid (Ile1153) decreased affinity for (−)-D888 from 10–20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (−)-D888 to approximately 9 μm, similar to the affinity of non-l-type Ca2+ channels for this drug. These results indicate that there are important determinants of phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor site at the interface between domains III and IV of the α1 subunit is presented.

Analysis of the dihydropyridine receptor site of L-type calcium channels by alanine-scanning mutagenesis.

The dihydropyridine Ca2+antagonist drugs used in the therapy of cardiovacular disorders inhibitl-type Ca2+ channels by binding to a single high affinity site. Photoaffinity labeling and analysis of mutant Ca2+ channels implicate the IIIS6 and IVS6 segments in high affinity binding. The amino acid residues that are required for high affinity binding of dihydropyridine Ca2+ channel antagonists were probed by alanine-scanning mutagenesis of the α1C subunit, transient expression in mammalian cells, and analysis by measurements of ligand binding and block of Ba2+ currents through expressed Ca2+ channels. Eleven amino acid residues in transmembrane segments IIIS6 and IVS6 were identified whose mutation reduced the affinity for the Ca2+ antagonist PN200-110 by 2–25-fold. Both amino acid residues conserved among Ca2+ channels and those specific to l-type Ca2+ channels were found to be required for high affinity dihydropyridine binding. In addition, mutation F1462A increased the affinity for the dihydropyridine Ca2+ antagonist PN200-110 by 416-fold with no effect on the affinity for the Ca2+ agonist Bay K8644. The residues in transmembrane segments IIIS6 and IVS6 that are required for high affinity binding are primarily aligned on single faces of these two α helices, supporting a “domain interface model” of dihydropyridine binding and action in which the IIIS6 and IVS6 interact to form a high affinity dihydropyridine receptor site on l-type Ca2+ channels.