80K-H as a New Ca2+ Sensor Regulating the Activity of the Epithelial Ca2+ Channel Transient Receptor Potential Cation Channel V5 (TRPV5)

Abstract

binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EFhand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EFhand pair reduced the TRPV5-mediated Ca 2؉ current and increased the TRPV5 sensitivity to intracellular Ca 2؉ , accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca 2؉ sensor controlling TRPV5 channel activity.The maintenance of the body Ca 2ϩ balance is of crucial importance for many vital physiological functions, including neuronal excitability, muscle contraction, and bone formation.The recent identification of two novel members of the transient receptor potential (TRP) 1 superfamily, TRPV5 and TRPV6, has provided new insight into the molecular mechanisms controlling the extracellular Ca 2ϩ balance (1-3). TRPV5 and TRPV6 are Ca 2ϩ -selective channels involved in transcellular Ca 2ϩ transport across vitamin D 3 -sensitive epithelia, such as kidney, intestine, and placenta (4, 5). Furthermore, these two channels possess unique functional characteristics among the highly heterogeneous group of TRP channels (6). They exhibit a constitutively activated Ca 2ϩ permeability, a high Ca 2ϩ selectivity, and a Ca 2ϩ -dependent feedback mechanism regulating channel activity (7,8).TRPV5 activity is controlled by various regulatory mechanisms ranging from long-term transcriptional modulations to short-term direct physical interactions with intracellular factors. TRPV5 is regulated at the transcriptional level by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), dietary Ca 2ϩ , and 17␤-estradiol (5, 9, 10). Previous studies have shown that these hormones and dietary Ca 2ϩ normalize the hypocalcemia in 25-hydroxyvitamin D 3 -1␣-hydroxylase knock-out mice, which is accompanied by an up-regulation of TRPV5 mRNA expression levels. Furthermore, TRPV5 modulation occurs at the post-translational level by controlling translocation of the channel from intracellular pools to the plasma membrane. Recently, the S100A10-annexin 2 complex was demonstrated to modulate the functional plasma membrane distribution of TRPV5 by direct association to the carboxyl (C)-terminal tail of the channel. Down-regulation of annexin 2 inhibited TRPV5-mediated currents in TRPV5-transfected cells (11). Finally, the terminal tails of the channel contain potential regulatory motifs, such as protein kinase C (PKC) phosphorylation sites, PDZ motifs, and ankyrin repeats (4). This suggests a regulatory role for the intracellular tails of TRPV5 in the (in)activation or the trafficking of the channel. In line with this observation, Niemeyer et al. (13) demonstrated that TRPV6 is competitively regulated by PKC and calmodulin.The aim of the present study was to identify ...

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

“…Inactivation of the EF-hand pair reduced the TRPV5-mediated Ca 2+ current and increased TRPV5 sensitivity to intracellular Ca 2+ , accelerating the feedback inhibition of channel activity. The membrane localization of 80K-H and TRPV5 was not altered, suggesting that 80K-H has a direct effect on TRPV5 activity (Gkika et al 2004). The 80K-H binding-site identified in the C-terminal tail of TRPV5 corresponds to the amino acid sequence MLERK, which is the same region where TRPV5 self-assembly can occur (Table 1; Chang et al 2004).…”

“…Deleting or mutating assembly domains could cause a change in tertiary structure and/or prevent the interaction with auxiliary proteins, thereby affecting channel trafficking and activity. For instance, the assembly domain in the C-terminal tail of TRPV5, the MLERK sequence, is also known to bind the protein 80K-H (Gkika et al 2004). Impaired trafficking observed in the MLERK-mutant of TRPV5 could be explained by the inability to interact with the necessary auxiliary proteins.…”

“…A study by Gkika et al demonstrated a functional role for 80K-H in the Ca 2+ -dependent regulation of TRPV5 channel activity (Gkika et al 2004). TRPV5 and 80K-H co-localized in 1,25 (OH) 2 D 3 -responsive epithelia, forming a heteromeric complex along the plasma membrane.…”

Regulation of TRPV5 and TRPV6 by associated proteins

“…Similarly, identification of the molecular players that associate with TRPV5 and TRPV6 could be pivotal in our understanding of the regulation of these channels. Until now, five regulatory proteins have been identified that associate with TRPV5 and/or TRPV6, i.e., S100A10-annexin 2, calmodulin (CaM), 80K-H, Na ϩ /H ϩ exchanger regulatory factor 2 (NHERF2), and Rab11a (17,22,35,40,46,61,62). S100A10/Annexin 2 S100A10 (also known as p11 or annexin 2 light chain) was identified as an auxiliary protein for TRPV5 and TRPV6 using a yeast two-hybrid system (62).…”

TRPV4-mediated channelopathies

“…In this regard, recent studies on other TRP channels indicate that membrane trafficking of TRPs are complex and different Rab-GTPase, dynamin, 80KH, annexins and kinesins might be involved. [74][75][76][77] Based on the structural information available and existing sequence homology among other TRPs, especially with TRPV members and the manner by which the surface expression of TRP channels are regulated, it can be speculated that proteins like Signal Transducing Adaptor Molecule (STAM), Hrs (which downregulate TRPP2 in C. elegans) and Recombinase Gene Activator (RGA, which regulates cell surface expression of TRPV2) may also be involved in the surface expression of TRPV4. [78][79][80] However, further detailed studies are needed to confirm if these proteins really interact and are involved in the surface expression of TRPV4.…”