Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS: We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

Salt stress-induced chloride flux: a study using transgenic Arabidopsis expressing a fluorescent anion probe.

Salt stress leads to massive accumulation of toxic levels of Na(+) and Cl(-) ions in plants. By using the recombinant fluorescent probe CLOMELEON, we demonstrate passive anion flux under salt stress. Chloride influx is restricted in the presence of divalent cations like Mg(2+) and Ca(2+), and completely blocked by La(3+). The amount but not the rate of the reported chloride uptake is independent from the kind of corresponding permeable cation (K(+) versus Na(+)), external pH and magnitude of osmotic stress. Cl(-) efflux however seems to involve stretch-activated transport. From the influence of Ca(2+) on reported changes of cytosolic anion concentrations, we speculate that transport mechanisms of Cl(-) and Na(+) might be thermodynamically coupled under saline conditions.

Proteolytic E-cadherin activation followed by solution NMR and X-ray crystallography.

Cellular adhesion by classical cadherins depends critically on the exact proteolytic removal of their N-terminal prosequences. In this combined solution NMR and X-ray crystallographic study, the consequences of propeptide cleavage of an epithelial cadherin construct (domains 1 and 2) were followed at atomic level. At low protein concentration, the N-terminal processing induces docking of the tryptophan-2 side-chain into a binding pocket on the same molecule. At high concentration, cleavage induces dimerization (KD=0.72 mM, k(off)=0.7 s(-1)) and concomitant intermolecular exchange of the betaA-strands and the tryptophan-2 side-chains. Thus, the cleavage represents the switch from a nonadhesive to the functional form of cadherin.

Peculiarities of aqueous amaranth starch suspensions.

Among the starches the granules from amaranth starch (a. hypochondriacus, amylopectin type) are singular because of their extremely small size of 1-3 microm and high uniformity. However, large spherical particles of 30-80 microm in diameter were observed from spray-dried amaranth starch by environmental scanning electron microscopy (ESEM) which exhibited a characteristic fine structure. After the mixture was stirred in cold water, the large particles disintegrate into small ones of 1-3 microm diameter. The particles from the stirred suspension were characterized by static and dynamic light scattering and viscometry. Almost the same diameters were found by the three techniques which were 1.76 times larger than those for the dry starch particles investigated by ESEM. The difference in size is explained by reversible uptake of water. A molar mass of M(w,granule) = 177 x 10(9) g/mol was measured for the granular particle. After dissolution in 0.5 N sodium hydroxide a molar mass M(w,amylopectin) = 104 x 10(6) g/mol of the amylopectin was obtained that agreed satisfactorily with that of waxy maize. Thus the granule consists on average of 1700 amylopectin molecules. Furthermore, rheological measurements were carried out with aqueous suspensions at room temperature. A shear gradient dependence was found for concentrations higher than 6% (w/v) of granules. At c > 19% reversible gel formation was observed with G'(omega) > G"(omega) and a plateau over 2 decades. The zero shear viscosity as a function of c[eta] shows behavior similar to glycogen and to latex particles. The granules, however, differ from common latex particles because of their capability to gel formation.