Outer hair cells underlie high frequency cochlear amplification in mammals. Fast somatic motility can be driven by voltage-dependent conformational changes in the motor protein, prestin, which resides exclusively within lateral plasma membrane of the cell. Yet, how a voltage-driven motor could contribute to high frequency amplification, despite the low-pass membrane filter of the cell, remains an enigma. The recent identification of prestin's Cl- sensitivity revealed an alternative mechanism in which intracellular Cl- fluctuations near prestin could influence the motor. We report the existence of a stretch-sensitive conductance within the lateral membrane that passes anions and cations and is gated at acoustic rates. The resultant intracellular Cl- oscillations near prestin may drive motor protein transitions, as evidenced by pronounced shifts in prestin's state-probability function along the voltage axis. The sensitivity of prestin's state probability to intracellular Cl- levels betokens a more complicated role for Cl- than a simple extrinsic voltage sensor. Instead, we suggest an allosteric modulation of prestin by Cl- and other anions. Finally, we hypothesize that prestin sensitivity to anion flux through the mechanically activated lateral membrane can provide a driving force that circumvents the membrane's low-pass filter, thus permitting amplification at high acoustic frequencies.

Background: TPC1 is an organellar NAADP-activated channel with unknown properties. Results: TPC1 functions as a pH-, Ca 2ϩ -, and voltage-regulated channel. Remarkably, activation of TPC1 by NAADP is dynamically regulated by the membrane potential. Conclusion:The properties of TPC1 can account for NAADP-evoked organellar Ca 2ϩ oscillations. Significance: These findings increase understanding of the organellar and receptor-evoked Ca 2ϩ signals.

Background: Evolutionary conserved Bax inhibitor-1 (BI-1) protects against ER stress-mediated apoptosis. Results: We identified a Ca 2ϩ -permeable channel pore in the C terminus of BI-1. Critical pore properties are an ␣-helical structure and two aspartate residues conserved among animals, but not among plants and yeast. Conclusion: C-terminal domain of BI-1 harbors a Ca 2ϩ -permeable channel pore. Significance: BI-1 has Ca 2ϩ channel properties likely relevant for its function in ER stress and apoptosis. Bax inhibitor-1 (BI-1) is a multitransmembrane domainspanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca 2؉homeostasis by acting as a Ca 2؉ -leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca 2؉ pore responsible for its Ca 2؉ -leak properties. We utilized a set of C-terminal peptides to screen for Ca 2؉ leak activity in unidirectional 45 Ca 2؉ -flux experiments and identified an ␣-helical 20-amino acid peptide causing Ca 2؉ leak from the ER. The Ca 2؉ leak was independent of endogenous ER Ca 2؉ -release channels or other Ca 2؉ -leak mechanisms, namely translocons and presenilins. The Ca 2؉ -permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca 2؉ -leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca 2؉ -leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca 2؉ -channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca 2؉ leak. Thus, we elucidated residues critically important for BI-1-mediated Ca 2؉ leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca2؉ -leak properties of BI-1 are an added function during evolution. leak from the ER (9, 10). BI-1 seems to be strongly evolutionarily conserved and BI-1 orthologs from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function (11). Besides this, other diverse functions of BI-1 have been described. BI-1 is a negative regulator of the ER-stress sensor (12), it interacts with G-actin and increases actin polymerization (13), enhances cancer metastasis by altering glucose metabolism and by activating a sodium-hydrogen exchanger (14), and it reduces production of reactive oxygen species through direct interaction with NADPH-P450 reductase (15), a member of the microsomal monooxygenase system.Recently, the role of BI-1 in Ca 2ϩ signaling has been further explored. The effect of BI-1 on cell death seems to involve changes in the amount of Ca 2ϩ that is releasable from intrace...

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