Shumin Bian

Interactions between β-Catenin and the HSlo Potassium Channel Regulates HSlo Surface Expression

Background The large conductance calcium-activated potassium channel alpha-subunit (Slo) is widely distributed throughout the body and plays an important role in a number of diseases. Prior work has shown that Slo, through its S10 region, interacts with β-catenin, a key component of the cytoskeleton framework and the Wnt signaling pathway. However, the physiological significance of this interaction was not clear. Methodology/Principal Findings Using a combination of proteomic and cell biology tools we show the existence of additional multiple binding sites in Slo, and explore in detail β-catenin interactions with the S10 region. We demonstrate that deletion of this region reduces Slo surface expression in HEK cells, which indicates that interaction with beta-catenin is important for Slo surface expression. This is confirmed by reduced expression of Slo in HEK cells and chicken (Gallus gallus domesticus leghorn white) hair cells treated with siRNA to β-catenin. HSlo reciprocally co-immunoprecipitates with β-catenin, indicating a stable binding between these two proteins, with the S10 deletion mutant having reduced binding with β-catenin. We also observed that mutations of the two putative GSK phosphorylation sites within the S10 region affect both the surface expression of Slo and the channels voltage and calcium sensitivities. Interestingly, expression of exogenous Slo in HEK cells inhibits β-catenin-dependent canonical Wnt signaling. Conclusions and Significance These studies identify for the first time a central role for β-catenin in mediating Slo surface expression. Additionally we show that Slo overexpression can lead to downregulation of Wnt signaling.

Prestin Surface Expression and Activity Are Augmented by Interaction with MAP1S, a Microtubule-associated Protein

Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Qsp) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Qsp in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Qsp in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.

A highly expressing Tet-inducible cell line recapitulates in situ developmental changes in prestin's Boltzmann characteristics and reveals early maturational events.

Prestin is the motor protein within the lateral membrane of outer hair cells (OHCs), and it is required for mammalian cochlear amplification. Expression of prestin precedes the onset of hearing in mice, and it has been suggested that prestin undergoes a functional maturation within the membrane coincident with the onset of hearing. We have developed a tetracycline-inducible prestin-expressing cell line that we have used to model prestins functional maturation. We used prestins voltage-dependent nonlinear charge movement (or nonlinear capacitance) as a test of function and correlated it to biochemical measures of prestin expressed on the cell surface. An initial stage of slow growth in charge density is accompanied by a rapid increase in our estimate of charge carried by an individual motor. A rapid growth in charge density follows and strongly correlates with an increasing ratio between an apparently larger and smaller monomer, suggesting that the latter exerts a dominant-negative effect on function. Finally, there is a gradual depolarizing shift in the voltage of peak capacitance, similar to that observed in developing OHCs. This inducible system offers many opportunities for detailed studies of prestin.

Expression, Purification and Functional Reconstitution of Slack Sodium-Activated Potassium Channels

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na+-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Real time measures of prestin charge and fluorescence during plasma membrane trafficking reveal sub-tetrameric activity.

Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional prestin is not an obligate tetramer.

Combinatorial cysteine mutagenesis reveals a critical intramonomer role for cysteines in prestin voltage sensing.

Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.

Current carried by the Slc26 family member prestin does not flow through the transporter pathway

Prestin in the lateral membrane of outer hair cells, is responsible for electromotility (EM) and a corresponding nonlinear capacitance (NLC). Prestin’s voltage sensitivity is influenced by intracellular chloride. A regulator of intracellular chloride is a stretch-sensitive, non-selective conductance within the lateral membrane, GmetL. We determine that prestin itself possesses a stretch-sensitive, non-selective conductance that is largest in the presence of thiocyanate ions. This conductance is independent of the anion transporter mechanism. Prestin has been modeled, based on structural data from related anion transporters (SLC26Dg and UraA), to have a 7 + 7 inverted repeat structure with anion transport initiated by chloride binding at the intracellular cleft. Mutation of residues that bind intracellular chloride, and salicylate treatment which prevents chloride binding, have no effect on thiocyanate conductance. In contrast, other mutations reduce the conductance while preserving NLC. When superimposed on prestin’s structure, the location of these mutations indicates that the ion permeation pathway lies between the core and gate ring of helices, distinct from the transporter pathway. The uncoupled current is reminiscent of an omega current in voltage-gated ion channels. We suggest that prestin itself is the main regulator of intracellular chloride concentration via a route distinct from its transporter pathway.

Evaluating Prestin's Changing Biophysical Attributes in Development Using a Tet‐Induced Cell Line

The motor protein prestin is a member of the SLC26 anion transporter family, and expressed in the lateral wall of OHCs. It is now widely recognized that prestin is required for mammalian cochlear amplification. Expression of prestin precedes the onset of hearing in mice and undergoes a functional maturation within the membrane coincident with the onset of hearing. We have developed several tetracycline‐inducible prestin expressing cell lines that duplicate prestins functional maturation in vivo. Thus, following induction there is an initial stage of increase in the charge carried by an individual motor (z) that accompanies a phase of slow growth in charge density. A plateau in z follows and is accompanied by rapid increase in charge density. The latter strongly correlates with an increasing ratio between an apparently larger and smaller monomer of prestin, suggesting that the latter exerts a dominant negative effect on function. Through the experimental period there is a progressive shift in the voltage o...