The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na(+), K(+), Ca(2+) and Mg(2+)) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (>or=10(7)MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M(r) 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca(2+) permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.

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of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family. Can. J. Biochem. 50,1282Biochem. 50, -1296.The kinetics of the reaction of nitrous acid at 4 O and pH 4.8 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the H O N 8 concentration showing that N28, was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pKvalues. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate sf nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by sptrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for a-amino groups, &-amino groups, and other residues in proteins were compared. The confornational state of the N-terminal residues in serine proteinases, as revealed from theit reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially a-amino groups and indole rings.A., et HOFMANN, T. Kinetics of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family. Can. J. Biochem. 50,1282Biochem. 50, -1296Biochem. 50, (1972.Nous avons ttudik le cinetique de la rtaction de l'acide nitreux a 4" et a pH 4.8 avec divers acides amints, des peptides et des protkines. La reaction avec I'ester mtthylk de l'isoleucine montre une dkpenbnce lintaire avec le c a r t de la concentration de HONO. La substance qui rkagit est donc le N203. Les constantes de vitesse de nitrosation de troisitme ordre des groupements amints primaires sont en relation avec leurs valeurs de pK. NOW les avons calculkes pour la concentration des composts non protonises pour donner les rkactivites intrinkques. La v i t e s~ de nitrosation de l'acttyltryptophanne en N-nitrosoacttyltryptophanne est fonction lintaire de la concentration de B'acide nitreux. Cette nitrosation suit donc un mkcanisme diffkrent. La rkaction de l'acide nitreux avec les rCsidus de tyrosine est examinte par spectrophotornetrie. La rtaction est nbgligeable comparte B celle des autres groupements. L'adtyl histidine et l'imidazsle ne rtagissent pas. Nous comparons la rkactivitt des groupements a-amints, e-aminb et celle d'autres rtsidus dans les proteines. Nous discutons en dktail l'ktat conformationnel des rtsidus N-terminaux des sCrine proteinases tel que montrk par leur rtactivitk. Nous concluons que I'acide nitreux rkagit de faqon prefkrentielle avec les rCsidus de '6s...

The reactivity of the α-amino group of isoleucine-16 of α-chymotrypsin towards nitrous acid at pH 4.0 and 0° is strongly dependent on ionic strength. Third-order deamination rate constants at low ionic strength (μ = 0.1 M) are 500–1000 times higher than those at high ionic strength (μ = 5.0 and 6.0 M) and are independent of the nature of the ions and the chymotrypsin concentration. Extrapolation to zero ionic strength of a plot of the logarithms of the constants against ionic strength leads to a value which is the same as that for the exposed α-amino group of the model compounds isoleucylvaline and valylvaline, and of the N-terminal isoleucine of pepsin. The deamination rate constant of the dipeptide valylvaline varies only two- to three-fold between ionic strength of 0.1 M and 6 M. The results suggest that the concept of a "buried" N-terminal as shown by X-ray analysis (carried out at pH 4.2 and ionic strength 9–11 M) requires modification; at low ionic strength (0.1 M) the reactivity of the N-terminal is only little below that of an exposed amino group, a fact which suggests that the amino group is much more available than shown by the X-ray analysis. The results are interpreted in terms of an effect of the ionic strength on the equilibrium between two conformational states of the enzyme.