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The maintenance of a differentiated chondrocyte phenotype is influenced by several factors of which signal transduction of extracellular stimuli through the cell membrane is of major interest. One important group of membrane-bound proteins which are involved in transmembrane signal transduction are ion channels. Human articular chondrocytes were obtained from osteoarthritic femoral condyles. Cells were released from the surrounding matrix and cultivated under standard conditions. We investigated gene expression of 12 members of the TRP ion channel family of freshly prepared (passage 0; P0) and in vitro propagated human articular chondrocytes (passage 2; P2) using conventional and real-time PCR (RT-PCR). In addition, the protein appearance of four TRP channels was demonstrated by immunofluorescence and western blotting. Chondrocyte differentiation was monitored by quantification of collagen type-II, type-I, and aggrecan gene expression. By conventional PCR, 8 channels could be detected, of which some displayed a heterogeneous PCR pattern. RT-PCR quantification revealed that TRPC1 was expressed on the same level in P0 and P2 chondrocytes while gene expression of TRPC3 and TRPC6 was elevated in passage 2 cells. TRPM5, TRPM7, and TRPV1 displayed an enhanced gene expression in freshly isolated chondrocytes. Immunofluorescence signal intensity of all four investigated TRP proteins was consistent with the corresponding gene expression data. In the present study, a correlation between the appearance of some members of the TRP ion channel family and the state of de-differentiation of osteoarthritic articular chondrocytes was shown. A possible direct involvement in the process of chondrocyte de-differentiation has to be investigated in further studies.

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.