We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4 Mutations to Met680 , which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca 2؉ permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys 675 , had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

A novel member of the transient receptor potential (Trp) family of ion channels, Trp12, was identified. The Trp12 mRNA is abundantly expressed in mouse kidney and encodes a protein of 871 amino acid residues. Trp12 transfected cells reveal an elevated cytosolic Ca 2+ and respond with a further increase of cytosolic Ca 2+ to perfusion with hypoosmotic solutions. The human orthologue of murine Trp12 was localized on a genomic clone derived from human chromosome 12. It is composed of 15 translated exons. The intron placement within that primary structure does not correlate with the previously postulated splice sites in transcripts encoding the stretch-inhibitable channel which shares a high degree of amino acid sequence identity with Trp12 and the vanilloid receptor type 1. ß

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The detection of changes in volume and osmolality is an essential function in vertebrate cells. A novel member of the transient receptor potential (trp) family of ion channels, which is sensitive to changes in cell volume, has been described recently. Heterologous expression of TRP12 in HEK cells resulted in the appearance of a swelling-activated cation current. The permeability sequence of this cation current for various monovalent cations, as determined from shifts in reversal potential upon extracellular cation substitution, was PK>PCs>PNa>PLi, corresponding to an Eisenman-IV sequence characteristic for a weak-field-strength site. Surprisingly, over-expression of this channel in HEK cells was accompanied by a dramatic down-regulation of the volume-regulated anion channel (VRAC), which is activated by cell swelling in non-transfected cells. In contrast to VRAC, TRP12 could not be activated at constant volume by a reduction of intracellular ionic strength or by intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate (GTPgammaS). The kinetic and pharmacological profile of VRAC and TRP12 currents were also different.

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The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca 2؉ transporterlike protein and Ca 2؉ transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca 2؉ -selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca 2؉ entry in electrically nonexcitable cells. Calcium is involved in a multitude of intracellular signal transduction mechanisms ranging from contraction to secretion. To achieve a high bandwidth of signal transmission with sometimes even opposing effects, cells need to control the spatio-temporal resolution of their Ca 2ϩ signals (1). In nonexcitable cells the rise in [Ca 2ϩ ] i 1 during stimulation of G-proteincoupled receptors or receptor tyrosine kinases is regulated in a complex fashion by Ca 2ϩ release from endogenous IP 3 -sensitive stores followed by store-operated Ca 2ϩ influx across the plasma membrane. One of the best characterized store-operated Ca 2ϩ entry pathways is the Ca 2ϩ release-activated Ca 2ϩ current, termed I CRAC (2, 3).The molecular identity of store-operated channels is a matter of debate. Several members of the TRP family, a group of Ca 2ϩ permeable channels related to the Drosophila melanogaster TRP gene product, have been implicated in store-dependent cation influx (4, 5). However, so far, none of the trp genes has been shown to encode channels with the ion-permeation properties and the sensitivity to store depleting agents of CRAC channels.The family of the trp genes in vertebrates can be divided into three subfamilies, based on similarities in the structures of the encoded proteins (6), the TRPC, the TRPM, and the TRPV subfamilies. Members of the TRPV subfamily appear to be regulated by physical or chemical stimuli such as heat, osmotic, or mechanical stress. The recently cloned human TRPV6 gene product (GenBank TM accession no. CAC20417), formerly called CaT-L or TRP8 (7, 8) or CaT1 (9), belongs into this group. Human TRPV6 channels (7), like rat TRPV6 (formerly called rat CaT1; Ref. 10) and rabbit TRPV5 channels (former ECaC for epithelial Ca 2ϩ channel; Ref. 11), show a high Ca 2ϩ selectivity, Ca 2ϩ and Na ϩ permeation properties indicative of an anomalous mole fraction behavior, and current-voltage relationships similar to CRAC channels.Recently these common features between TRPV6 and CRAC channels were demonstrated by two groups (12, 13). In addition Voets et al. (13) also revealed several differences between I CRAC in RBL cells and TRPV6-mediated Ca 2ϩ currents when expressed in HEK cells, including insensitivity of TRPV6 channels to store depletion and the effect of intracellular Mg 2ϩ , which causes voltage-dependent block of TRPV6, but not of CRAC channels. In contrast, Yue et al. (12) demonstrated activation of TRPV6 channels in CHO cells by depletion of calcium stores with...

⌬695-725 , which lacks the C-terminal domain required for Ca 2؉ -calmodulin binding, does not form constitutive active channels, whereas the human TRPV6 D542A , carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.