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1. 1. Radioligand binding and 45Ca2+ uptake measurements quantitated ion channel numbers and properties in brain membranes from Fisher 344 rats at 6, 12, 18 and 30 months of age. 2. 2. Decreases in 1,4-dihydropyridine density occurred in striatum, hippocampus and cortex with a decreased affinity. 3. 3. Decreases in w-conotoxin binding occurred in hippocampus and striatum with an increase in affinity. 4. 4. K+ depolarization-mediated 45Ca2+ uptake decreased only in striatum at 18 months. 5. 5. Decreases in glibenclamide binding occurred in cortex and cerebellum at 12–30 months. 6. 6. No changes in 1,4-dihydropyridine binding occurred with age in heart, but glibenclamide binding density was significantly decreased at 30 months.

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.

Administration of 3,3'-iminodipropionitrile (IDPN) (1 g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN-induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200-110 binding sites (Bmax) in whole brains of IDPN-treated mice. Similarly, the K(+)-depolarization-dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 +/- 7 fmol/mg to 237 +/- 31 fmol/mg in control and treated groups, respectively) of PN 200-110 binding to the striatum without change of KD value (38 +/- 4.7 pM versus 33 +/- 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 +/- 10.1 pM to 45 +/- 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 +/- 51 fmol/mg versus 485 +/- 41 fmol/mg) of PN 200-110 binding to the cortex. IDPN did not alter omega-conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment as inhibited by L-type calcium channel antagonists and this may be associated with the observed increase in striatal L-type calcium channels.

We have used whole cell patch clamp procedures to investigate the inhibition of L-type calcium channel currents in guinea pig ventricular cells by the permanently charged dihydropyridine (DHP)compound UK-118,434-05 (quaternary amlodipine, QA). The location of the charge group of this drug molecule is approximately three times closer to the active DHP moiety than is the case for SDZ-207-180, the only other previously-investigated quaternary DHP molecule. Like SDZ-207-180, QA inhibits channel activity only by external application, consistent with an externally, but not internally, accessible binding site, and once blocked, channels do not recover availability by membrane hyperpolarization independent of extracellular pH. However inhibition by QA occurs at roughly 20 x lower potency than comparable inhibition by SDZ-207-180. Low affinity binding to the DHP binding site was confirmed directly with radioligand binding. The permanently charged amlodipine derivative inhibited radioligand DHP binding in partially purified rabbit skeletal muscle transverse tubule membranes with a pseudo-Hill slope close to unity and an IC50 value of 4.2 +/- 0.6 microM. These results indicate that the characteristically slow pharmacokinetics of tertiary amlodipine are due to the unusually stable inhibition of L-channels caused by the ionized fraction of drug molecules. Furthermore, because the distance between the ionized head group and the DHP moiety is so short, the low affinity binding and channel inhibition by QA suggests that the DHP binding site is not on the extracellular domain of the L-channel alpha 1 subunit, but instead must reside within the bilayer or channel pore at a location closer to the extracellular rather than the intracellular face of the membrane.

Publisher Summary Drugs that regulate Ca 2+ influx via voltage-gated Ca 2+ channels are called calcium antagonists or calcium-channel blockers. Phenylalkylamines, benzothiazepines, and the 1,4-dihydropyridines (DHPs) are drugs that bind to distinct but allosterically coupled receptors on the channel protein. DHPs bind to the channel with highest affinity and specificity. Calcium-channel antagonists are unique drugs; they have made major contributions to clinical medicine and the basic science of calcium-channel proteins. Because of the multiple roles of calcium ions in cell function, communication, and biochemical regulation, the control of calcium entry by calcium-channel antagonists has given therapeutic tools for a wide variety of clinical disorders. The high degree of selectivity and great sensitivity of calcium-channel antagonists for the L-type channel allow the cloning and molecular fingerprinting of the channel protein. Calcium-channel antagonists, in general, and the DHP derivatives, in particular, regulate calcium influx by modulating channel gating. This chapter presents a method to explain the voltage-dependent modulation of L-type calcium-channel activity by DHP derivatives. It discusses DHP modulation of recombinant channel activity.