William A. Horne

Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel alpha 1 subunit known as alpha 1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature 350, 398-402] is generally assumed to encode the P-type Ca2+ channel. However, we find that alpha 1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins omega-Aga-IVA and omega-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron 11, 291-303]. Thus, alpha 1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another alpha 1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Horne W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787-3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Horne W. (1993) Nature 363, 455-458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or omega-Aga-IVA. Doe-1 current is reduced by omega-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxins potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to alpha 1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr. 19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular diversity of voltage-dependent Ca2+ channels

Voltage-dependent Ca2+ channels regulate Ca2+ entry and thereby contribute to Ca2+ signalling in many cells. Functional studies have uncovered several types of Ca2+ channel, distinguished by pharmacology, electrophysiology and tissue localization. More recently, molecular cloning has revealed an even greater diversity among Ca2+ channels, arising from multiple genes and alternative splicing. L-type, dihydropyridine-sensitive Ca2+ channels have been the most extensively characterized to date. Recently, Numas group has reported the cloning and expression of a dihydropyridine-insensitive Ca2+ channel from brain that most closely resembles the P-type channel described by Llinas and colleagues. These results contribute to rapidly growing knowledge about molecular determinants of Ca2+ channel diversity.

Pharmacokinetics of single-dose oral pregabalin administration in normal dogs

OBJECTIVE To describe the pharmacokinetics of pregabalin in normal dogs after a single oral dose. STUDY DESIGN Prospective experiment. ANIMALS Six adult Labrador/Greyhound dogs (four females and two males) aged 2.6 (2.6-5.6) years old (median and range) weighing 33.4 (26.8-42.1) kg. METHODS After jugular vein catheterization, the dogs received a single oral dose of pregabalin ( approximately 4 mg kg(-1)). Blood samples were collected at: 0 (before drug administration), 15 and 30 minutes and at 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 36 hours after drug administration. Plasma pregabalin concentration was measured by HPLC. Noncompartmental analysis was used to estimate pharmacokinetic variables. RESULTS No adverse effects were observed. The median (range) pharmacokinetic parameters were: Area under the curve from time 0 to 36 hours = 81.8 (56.5-92.1) microg hour mL(-1); absorption half-life = 0.38 (0.25-1.11) hours; elimination half-life = 6.90 (6.21-7.40) hours; time over 2.8 microg mL(-1) (the presumed minimal effective concentration) = 11.11 (6.97-14.47) hours; maximal plasma concentration (C(max)) = 7.15 (4.6-7.9) microg mL(-1); time for C(max) to occur = 1.5 (1.0-4.0) hours. Assuming an 8-hour dosing interval, predicted minimal, average, and maximal steady state plasma concentrations were 6.5 (4.8-8.1), 8.8 (7.3-10.9), and 13.0 (8.8-15.2) microg mL(-1). The corresponding values assuming a 12-hour interval were 3.8 (2.4-4.8), 6.8 (4.9-7.9), and 10.1 (6.6-11.6) microg mL(-1). CONCLUSIONS AND CLINICAL RELEVANCE Pregabalin 4 mg kg(-1) PO produces plasma concentrations within the extrapolated therapeutic range from humans for sufficient time to suggest that a twice daily dosing regime would be adequate. Further study of the drugs safety and efficacy for the treatment of neuropathic pain and seizures in dogs is warranted.

Alternative Splicing of the Ca2+ Channel β4 Subunit Confers Specificity for Gabapentin Inhibition of Cav2.1 Trafficking

Gabapentin is well established as an effective treatment for neuropathic pain; however, little is known about its mechanism of action. It binds with high affinity to Ca2+ channel α2δ subunits that are expressed in dorsal root ganglia. Mutation of a single α2δ amino acid, R217A, eliminates both gabapentin binding and analgesic efficacy. Gabapentin does not seem to have direct Ca2+ channel blocking properties but does affect overall levels of Ca2+ channel surface expression in some circumstances. In this report, we examined gabapentin effects on trafficking and voltage-dependent gating properties of recombinant Cav2.1 Ca2+ channel complexes transiently expressed in Xenopus laevis oocytes. We also determined electrophysiologically whether gabapentin causes displacement of β subunits from Cav2.1 complexes. Our principal findings are as follows: 1) gabapentin inhibits trafficking of recombinant Cav2.1 Ca2+ channels in X. laevis oocytes; 2) gabapentin inhibition occurs in the presence of the Ca2+ channel β4a subunit but not in the presence of β4b; 3) gabapentin does not affect Cav2.1 voltage-dependent gating parameters; 4) inhibition of Cav2.1trafficking is highly dependent on β-subunit concentration; and 5) gabapentin inhibition of Cav2.1 trafficking can be reversed by the α2δ R217A mutation. Overall, our results suggest that gabapentin reduces the number of β4a-bound Cav2.1 complexes that are successfully trafficked to the plasma membrane. This mechanism may help to explain why gabapentin is both effective and selective in the treatment of neuropathic pain states that involve up-regulation of α2δ subunits.

Ca2+ channel-independent requirement for MAGUK family CACNB4 genes in initiation of zebrafish epiboly

CACNB genes encode membrane-associated guanylate kinase (MAGUK) proteins once thought to function exclusively as auxiliary β subunits in assembly and gating of voltage-gated Ca2+ channels. Here, we report that zygotic deficiency of zebrafish β4 protein blocks initiation of epiboly, the first morphogenetic movement of teleost embryos. Reduced β4 function in the yolk syncytial layer (YSL) leads to abnormal division and dispersal of yolk syncytial nuclei, blastoderm retraction, and death, effects highly similar to microtubule disruption by nocodazole. Epiboly is restored by coinjection of human β4 cRNA or, surprisingly, by mutant cRNA encoding β4 subunits incapable of binding to Ca2+ channel α1 subunits. This study defines a YSL-driven zygotic mechanism essential for epiboly initiation and reveals a Ca2+ channel-independent β4 protein function potentially involving the cytoskeleton.

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.