Biomedical subjects

Tim T Lambers

Publications and source records attributed to Tim T Lambers.

4 recordsLinked to original sources

PubMed2006

Extracellular pH dynamically controls cell surface delivery of functional TRPV5 channels.

Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca(2+)-selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca(2+)-transporting cells, contributing significantly to the overall Ca(2+) balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, (45)Ca(2+) uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular "kiss and linger" interactions with the plasma membrane.

Animals↗

PubMed2006

RGS2 inhibits the epithelial Ca2+ channel TRPV6.

The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) entry pathway in the process of active Ca(2+) (re)absorption. By yeast two-hybrid and glutathione S-transferase pulldown analysis we identified RGS2 as a novel TRPV6-associated protein. RGS proteins determine the inactivation kinetics of heterotrimeric G-protein-coupled receptor (GPCR) signaling by regulating the GTPase activity of G(alpha) subunits. Here we demonstrate that TRPV6 interacts with the NH(2)-terminal domain of RGS2 in a Ca(2+)-independent fashion and that overexpression of RGS2 reduces the Na(+) and Ca(2+) current of TRPV6 but not that of TRPV5-transfected human embryonic kidney 293 (HEK293) cells. In contrast, overexpression of the deletion mutant DeltaN-RGS2, lacking the NH(2)-terminal domain of RGS2, in TRPV6-expressing HEK293 cells did not show this inhibition. Furthermore, cell surface biotinylation indicated that the inhibitory effect of RGS2 on TRPV6 activity is not mediated by differences in trafficking or retrieval of TRPV6 from the plasma membrane. This effect probably results from the direct interaction between RGS2 and TRPV6, affecting the gating properties of the channel. Finally, the scaffolding protein spinophilin, shown to recruit RGS2 and regulate GPCR-signaling via G(alpha), did not affect RGS2 binding and electrophysiological properties of TRPV6, indicating a GPCR-independent mechanism of TRPV6 regulation by RGS2.

Animals↗

PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗

PubMed2004

Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin.

TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) as well as the Na(+) current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca(2+)-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini (1-8-14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.

Amino Acid Sequence↗