Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties.

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.

Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling.

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.

Store-operated cation channels in the heart and cells of the cardiovascular system.

In many nonexcitable cells, activation of phospholipase C (PLC)-linked receptors results in a release of Ca(2+) from intracellular stores followed by a transmembrane Ca(2+) entry. This Ca(2+) entry underlies the sustained phase of [Ca(2+)](i) increase, is important for various cellular functions including gene expression, secretion and cell proliferation, and is supported by agonist-activated Ca(2+)-permeable ion channels. Ca(2+)-permeable channels which are activated by store depletion and which are therefore referred to as store- operated channels or SOCs form a major pathway for agonist-induced Ca(2+) influx. So far, the molecular structures of these channels have not been identified. Potential candidates are encoded by members of the TRP family, a class of ion channels initially discovered in Drosophila and involved in the PLC-dependent transduction of visual stimuli. Here, we review recent evidence that agonist-induced Ca(2+) influx and especially SOCs are present in different cell types of the heart and of the cardiovascular system and compare these findings with the possible functions and tissue-specific expression of mammalian TRP proteins.