Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeletons, and the formation and disassembly of cell-substratum adhesions. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily 'stuck' to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

The crucial role of L-type Ca2+ channels in the initiation of cardiac and smooth muscle contraction has made them major therapeutic targets for the treatment of cardiovascular disease. L-type channels share a common pharmacological profile, including high-affinity voltage- and frequency-dependent block by the phenylalkylamines, the benz(othi)azepines, and the dihydropyridines. These drugs are thought to bind to three separate receptor sites on L-type Ca2+ channels that are allosterically linked. Results from different experimental approaches implicate the IIIS5, IIIS6, and IVS6 transmembrane segments of the alpha 1 subunits of L-type Ca2+ channels in binding of all three classes of drugs. Site-directed mutagenesis has identified single amino acid residues within the IIIS5, IIIS6, and IVS6 transmembrane segments that are required for high-affinity binding of phenylalkylamines and/or dihydropyridines, providing further support for identification of these transmembrane segments as critical elements of the receptor sites for these two classes of drugs. The close proximity of the receptor sites for phenylalkylamines, benz(othi)azepines, and dihydropyridines raises the possibility that individual amino acid residues may be required for high-affinity binding of more than one of these ligands. Therefore, we suggest that phenylalkylamines and dihydropyridines bind to different faces of the IIIS6 and IVS6 transmembrane segments and, in some cases, bind to opposite sides of the side chains of the same amino acid residues. The results support the domain interface model for binding and channel modulation by these three classes of drugs.

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.

The high affinity phenylalkylamine (؊)D888 blocks ion currents through L-type Ca 2؉ channels containing the ␣ 1C subunit with an apparent K d of 50 nM, but N-type Ca 2؉ channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher K d value of 5 M. L-type Ca 2؉ channels containing ␣ 1C 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 ␣ 1C . Ca 2؉ 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 Ca 2؉ 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 ␣-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.Voltage-gated Ca 2ϩ channels constitute a family of integral plasma membrane proteins that form highly selective, calciumconducting pores upon membrane depolarization and thereby couple cell surface electrical signals to intracellular events such as contraction, secretion, and protein phosphorylation (reviewed in Refs. 1 and 2). The pore-forming ␣ 1 subunits of voltage-gated Ca 2ϩ channels consist of four homologous domains (I-IV), each containing six putative transmembrane segments (S1-S6) (3). Voltage-gated Ca 2ϩ channels are blocked by phenylalkylamines, which are thought to bind within the intracellular mouth of the ion-conducting pore (4). Block of L-type Ca 2ϩ channels in cardiac and smooth muscle by verapamil and related phenylalkylamines is an important therapy for hypertension, cardiac arrhythmias, and angina pectoris (5). In contrast, N-type Ca 2ϩ channels in neurons are relatively insensitive to block by these drugs.The phenylalkylamine (Ϫ)D888 (desmethoxyverapamil) binds to L-type Ca 2ϩ channels with high affinity (6) and potently blocks L-type currents (7). Photoaffinity labeling of purified skeletal muscle L-type Ca 2ϩ channels with the high affinity phenylalkylamine [N-methyl-3 H]LU49888 (8) resulted in highly selective derivatization of a peptide containing transmembrane segment S6 in domain IV (IVS6) (9). Several lines of evidence have also implicated the S6 segment of K ϩ channels (10 -13) and the IVS6 segment of Na ϩ channels (14) as components of the binding sites for intracellular pore-blocking drugs. These findings led us to investigate the role of segment IVS6 in high affinity phenylalkylamine block of L-type Ca 2ϩ channels. We report here that three amino acid residues in segment IVS6, Tyr-1463, Ala-1467, and Ile-1470, are required for high affinity block of L-type Ca 2ϩ channels by the phenylalkylamine (Ϫ)D888...

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