Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

Structural insight into TRPV5 channel function and modulation.

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

TRPV5 in renal tubular calcium handling and its potential relevance for nephrolithiasis.

Nephrolithiasis or renal stone disease is an increasingly common problem, and its relatively high recurrence rate demands better treatment options. The majority of patients with nephrolithiasis have stones that contain calcium (Ca2+), which develop upon "supersaturation" of the urine with insoluble Ca2+ salts; hence processes that influence the delivery and renal handling of Ca2+ may influence stone formation. Idiopathic hypercalciuria is indeed frequently observed in patients with kidney stones that contain Ca2+. Genetic screens of nephrolithiasis determinants have identified an increasing number of gene candidates, most of which are involved in renal Ca2+ handling. This review provides an outline of the current knowledge regarding genetics of nephrolithiasis and will mainly focus on the epithelial Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), an important player in Ca2+ homeostasis. Being a member of the TRP family of ion channels, TRPV5 is currently part of a revolution in structural biology. Recent technological breakthroughs in the cryo-electron microscopy field, combined with improvements in biochemical sample preparation, have resulted in high-resolution 3-dimensional structural models of integral membrane proteins, including TRPV5. These models currently are being used to explore the proteins' structure-function relationship, elucidate the molecular mechanisms of channel regulation, and study the putative effects of disease variants. Combined with other multidisciplinary approaches, this approach may open an avenue toward better understanding of the pathophysiological mechanisms involved in hypercalciuria and stone formation, and ultimately it may facilitate prevention of stone recurrence through the development of effective drugs.

TRP channels in calcium homeostasis: from hormonal control to structure-function relationship of TRPV5 and TRPV6.

Maintaining plasma calcium levels within a narrow range is of vital importance for many physiological functions. Therefore, calcium transport processes in the intestine, bone and kidney are tightly regulated to fine-tune the rate of absorption, storage and excretion. The TRPV5 and TRPV6 calcium channels are viewed as the gatekeepers of epithelial calcium transport. Several calciotropic hormones control the channels at the level of transcription, membrane expression, and function. Recent technological advances have provided the first near-atomic resolution structural models of several TRPV channels, allowing insight into their architecture. While this field is still in its infancy, it has increased our understanding of molecular channel regulation and holds great promise for future structure-function studies of these ion channels. This review will summarize the mechanisms that control the systemic calcium balance, as well as extrapolate structural views to the molecular functioning of TRPV5/6 channels in epithelial calcium transport.

Interspecies differences in PTH-mediated PKA phosphorylation of the epithelial calcium channel TRPV5.

The epithelial calcium (Ca2+) channel TRPV5 (transient receptor potential vanilloid 5) is expressed in the distal convoluted tubule of the kidney and facilitates active Ca2+ reabsorption. This process is instrumental for the maintenance of Ca2+ homeostasis. Therefore, all aspects of TRPV5 function are tightly regulated by the calciotropic parathyroid hormone (PTH). Rabbit (rb)TRPV5 channel activity was shown to be stimulated upon PTH-mediated protein kinase A (PKA) phosphorylation. Since there is incomplete conservation of the PKA consensus motif (RR/QxT) across species, the aim of this study was to extend these findings to humans and characterize the expression and function of human (h)TRPV5. Functional differences between rbTRPV5 and hTRPV5 upon PTH stimulation were investigated using 45Ca2+ uptake assays, Fura-2 Ca2+ imaging, and cell surface biotinylation. While PTH treatment enhanced rbTRPV5 channel activity, it did not stimulate hTRPV5 activity. Mutation of the human RQxT motif into rabbit RRxT (hTRPV5 Q706R) partially restored the sensitivity to PTH. An ancestral sequence reconstruction of TRPV5 orthologues demonstrated that the change in the RRxT motif coincides with the creation of another putative PKA motif (RGAS to RRAS) in the amino terminus of hTRPV5. Interestingly, a constitutively phosphorylated hTRPV5 mutant (hTRPV5 S141D) displayed significantly decreased channel function, while its plasma membrane abundance was increased. Taken together, PTH-mediated stimulation of TRPV5, via PKA, is not conserved in humans. Our data suggest that PTH regulation of TRPV5 is altered in humans, an important observation for future studies that may add to new concepts on the role of PTH in renal Ca2+ handling.

Title: Jealous protons sour another happy marriage; the story of how TRPV5 and PI(4,5)P2 split up.

TRPV5 is a highly selective calcium channel that finetunes urinary calcium excretion by reabsorbing calcium from the pro-urine. New structural findings show how PTH and pH control TRPV5 activity by altering the binding of endogenous ligands calmodulin and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).

High-resolution structures of transient receptor potential vanilloid channels: Unveiling a functionally diverse group of ion channels.

Transient receptor potential vanilloid (TRPV) channels are part of the superfamily of TRP ion channels and play important roles in widespread physiological processes including both neuronal and non-neuronal pathways. Various diseases such as skeletal abnormalities, chronic pain, and cancer are associated with dysfunction of a TRPV channel. In order to obtain full understanding of disease pathogenesis and create opportunities for therapeutic intervention, it is essential to unravel how these channels function at a molecular level. In the past decade, incredible progress has been made in biochemical sample preparation of large membrane proteins and structural biology techniques, including cryo-electron microscopy. This has resulted in high resolution structures of all TRPV channels, which has provided novel insights into the molecular mechanisms of channel gating and regulation that will be summarized in this review.