Claudia Trost

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.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca2+ entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry thatTRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only otherTRP transcript found at considerable levels wasTRP1, whereas TRP2, TRP3, TRP5(CCE2), andTRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca2+ (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Pain Perception in Mice Lacking the β3 Subunit of Voltage-activated Calcium Channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the α1B subunit (N-type). The accessory β3 subunits of calcium channels are preferentially associated with the α1B subunit in neurones. Here we show that deletion of the β3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cordin vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of β3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (α1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

CA2+‐dependent interaction of the trpl cation channel and calmodulin

The transient receptor potential‐like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential‐like fusion proteins to characterize calmodulin binding regions in the transient receptor potential‐like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C‐terminus of the transient receptor potential‐like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710–I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent K i values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 μM (calmodulin binding site 1) and 3.3 μM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+‐calmodulin‐dependent mechanism contributes to transient receptor potential‐like cation channel modulation when expressed in CHO cells.