Showing papers by "Franz Hofmann published in 1997"

Regional Expression and Cellular Localization of the α1 and β Subunit of High Voltage-Activated Calcium Channels in Rat Brain

Abstract: The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different α1 and four different β subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The β subunit-specific antibodies identified the presence of β1a in skeletal muscle; β2 in heart; and β2, β3, and β4 in brain. The β3protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The β4protein was also widely expressed, most prominently in the cerebellum. β2 protein was expressed at only low levels. In situ hybridization with β subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the α1A, α1B, α1C, and α1D subunits showed a broad distribution of α1A and α1B transcripts, whereas the expression level of α1C and α1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that β4 may associate predominantly, but probably not exclusively, with the α1Asubunit, and β3 with the α1B subunit. In certain brain areas such as the habenula, the β3 subunit may associate with other α1subunits too. Furthermore, the β2 subunit may form complexes with different α1 subunits in brain and cardiac muscle. These results demonstrate that a given β subunit may associate with different α1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Alternatively Spliced IS6 Segments of the α1C Gene Determine the Tissue-Specific Dihydropyridine Sensitivity of Cardiac and Vascular Smooth Muscle L-Type Ca2+ Channels

Abstract: Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their α1 subunit, which binds the DHPs, is derived from the same gene. This α1C gene gives rise to several splice variants, among which the α1C-b variant is affected by lower concentrations of nisoldipine than the α1C-a variant. Functional expression of chimeras of α1C-a and α1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the α1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the α1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 α1C-a and IS6 α1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities...

Transfer of the High Affinity Dihydropyridine Sensitivity from L-Type To Non-L-Type Calcium Channel

Abstract: To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive α1C and DHP-insensitive α1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the α1C sequence completely abolished the inhibition and stimulation of IBaby the antagonist (+)-isradipine and agonist (−)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the α1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the α1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated α1E channel was stimulated 2.8-fold by 1 μm Bay K 8644 and blocked by isradipine with an IC50 value of 60 nm. These results show that mutation of Thr1061 in the α1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the α1E sequence.

Identification and functional characterization of a calcium channel gamma subunit.

Abstract: A positive selection technique was used to identify novel auxiliary calcium channel subunits that are similar to the skeletal muscle γ subunit. A new rat γ subunit cDNA was found, which was highly expressed in skeletal muscle tissue and was detected by RT-PCR in cardiac tissue. The 223-amino-acid-protein shares 84% and 79% identity, respectively, with the human and rabbit skeletal muscle subunits. Northern blot analysis revealed a single transcript of 1.5 kb in rat skeletal muscle, but not in cardiac tissue. Transient coexpression with the cardiac calcium channel complex demonstrated that the γ subunit shifted the inactivation curve to negative potentials and accelerated current inactivation without changing other voltage-dependent properties of the channel.