Distribution of voltage gated calcium channel ß subunits in the mouse retina.

Voltage gated calcium channels (VGCCs) are essential to neuronal excitation and signal transduction. They are multimeric in structure and comprised of an alpha subunit that functions as a calcium pore and two additional subunits: an alpha2delta subunit and a cytoplasmic beta subunit. To better understand the role of VGCCs in the retina we used immunohistochemical methods to determine the distribution of VGCC ß subunits in normal and mutant mice. To verify the specificity of each antibody and to examine the potential for subunit redistribution when beta subunit expression is perturbed, we used 4 mutant mouse lines that each lack a specific ß subunit isoform (ß(1)-ß(4)). We found the ß(1) subunit distributed on cell bodies in the inner nuclear layer (INL) and on processes within both the inner and outer limiting membrane; the ß(2) subunit localized to the outer plexiform layer (OPL) and inner plexiform layer (IPL); the ß(3) subunit was localized to three narrow and distinct bands within the IPL; the ß(4) subunit was localized to three diffuse bands within the IPL. Loss of one ß subunit affected labeling intensity but not general distribution patterns of other ß subunits. It is likely that VGCCs critical for retinal signal transmission are comprised of the ß(2) subunit in the OPL and any of the 4 ß subunits in the IPL. Our results suggest that within the OPL the α(1F) subunit pairs predominantly with the ß(2) subunit while within the IPL it may pair with either any ß subunit.

Molecular heterogeneity of calcium channel beta-subunits in canine and human heart: evidence for differential subcellular localization.

Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Ca(v)beta genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca(v)beta isoforms. A total of 18 different Ca(v)beta isoforms were detected in both canine and human ventricles including splice variants from all four Ca(v)beta genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca(v)beta subunit genes are expressed at the protein level, and the Ca(v)beta subunits show differential subcellular localization with Ca(v)beta1b, Ca(v)beta2, and Ca(v)beta3 predominantly localized to the T-tubule sarcolemma, whereas Ca(v)beta1a and Ca(v)beta4 are more prevalent in the surface sarcolemma. Coexpression of the novel Ca(v)beta2c subunits (Ca(v)beta(2cN1), Ca(v)beta(2cN2), Ca(v)beta(2cN4)) with the pore-forming alpha1C (Ca(v)1.2) and Ca(v)alpha2delta subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca(v)beta2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca(v)beta subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

Low-voltage-activated ("T-Type") calcium channels in review.

The past 5 years has witnessed an advance in our understanding of alpha1G (Cav3.1), alpha1H (Cav3.2), and alpha11 (Car3.3), the pore-forming subunits of T-type or low-voltage-activated calcium channels (LVAs). LVAs differ in their localization and molecular, biophysical, and biochemical properties, but all conduct a transient calcium current in a variety of cells. T-type currents mediate a number of physiological functions in developing and mature cells, and are implicated in neural and cardiovascular diseases. Hampered by a lack of selective antagonists, characterization of T-type channels has come from recombinant channel studies and use of pharmacological and electrophysiological methods to isolate endogenous T-type currents. The surprising heterogeneity in T-type currents likely results from differences in LVA molecular composition, temporal and spatial localization, and association with modulatory molecules. A fundamental knowledge of LVA biochemical properties, including the molecular composition of endogenous LVAs and spatial and temporal characterization of protein expression, is necessary to elucidate mechanisms for regulation of expression and function in normal and diseased cells.

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.