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Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane.

ABSTRACT: The mechano-electrical transduction (MET) channel of the inner ear receptor cells, termed hair cells, is a protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to form a multi-protein complex with other MET channel proteins, such as PCDH15, TMIE, and TMC1. Despite localizing to the plasma membrane of stereocilia, the mechanosensing organelles of hair cells, LHFPL5 requires its binding partner within the MET complex, PCDH15, to localize to the stereocilia tips in hair cells and to the plasma membrane in heterologous cells. Using the Aquaporin 3-tGFP reporter (AGR) for plasma membrane localization, we found that a region within extracellular loop 1, which interacts with PCDH15, precludes the trafficking of AGR reporter to the plasma membrane in heterologous cell lines. Our results suggest that the presence of protein partners may mask endoplasmic reticulum retention regions or enable the proper folding and trafficking of the MET complex components, to facilitate expression of the MET complex at the stereocilia membrane.

SUBMITTER: Soler DC

PROVIDER: S-EPMC9925724 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane.

Soler David C DC Ballesteros Angela A Sloan Andrew E AE McCormick Thomas S TS Stepanyan Ruben R

Scientific reports 20230213 1

The mechano-electrical transduction (MET) channel of the inner ear receptor cells, termed hair cells, is a protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to form a multi-protein complex with other MET channel proteins, such as PCDH15, TMIE, and TMC1. ...[more]

PMID: 36781873

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Project description:As a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a unique Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. TRPV5-mediated Ca2+ entry into the cell is controlled by a negative feedback mechanism, in which calmodulin (CaM) blocks the TRPV5 pore upon Ca2+ binding. Combining microscopy techniques and biochemical assays, the present study uncovered an auxiliary role for CaM in the regulation of human (h)TRPV5 intracellular trafficking. Overexpressed hTRPV5 was mainly localised to the endoplasmic reticulum (ER) and associated with peripheral ER tubules. Limiting expression using the HEK293 TET-off system revealed that hTRPV5 trafficked through the endocytic recycling pathway. CaM co-localised with hTRPV5 at intracellular sites and overexpression of CaM slowed hTRPV5 exit from the ER. In accordance, CaM binding-disrupting truncations of the TRPV5 C-terminus (698X) or knockdown of endogenous CaM by small interfering RNA resulted in an increased fraction of TRPV5 that localised to the plasma membrane. hTRPV5 expressing cells had an increased intracellular Ca2+ concentration upon knockdown of CaM. The protein abundance of the Ca2+ impermeable hTRPV5-D542 mutant is also regulated by CaM, which suggests that the mode of action is independent of disrupted intracellular calcium concentrations. In conclusion, our study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, modulating TRPV5 intracellular trafficking. KEY POINTS: The renal Ca2+ channel TRPV5 is a crucial player in maintenance of the body's Ca2+ homeostasis. Ca2+ transport through TRPV5 is controlled by single channel activity, as well as TRPV5 plasma membrane abundance. Calmodulin (CaM) co-localised with TRPV5 at intracellular sites and retained TRPV5 in the endoplasmic reticulum. Disrupted CaM-TRPV5 binding or knockdown of endogenous CaM by small interfering RNA (siRNA) resulted in an increased TRPV5 plasma membrane abundance. Knockdown of endogenous CaM by siRNA resulted in increased intracellular Ca2+ concentrations. The regulation of TRPV5 trafficking by CaM is independent of the effect of CaM on intracellular Ca2+ concentrations. This study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, next to its ability to directly block the TRPV5 channel pore, by modulating TRPV5 trafficking in the secretory pathway.

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Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane.

Publications

Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane.

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