Volume-regulated anion channels (VRACs) are central to cell volume regulation. Recently identified as hetero-hexamers formed by LRRC8 proteins, their activation mechanism remains elusive. Here, we measured Förster resonance energy transfer (FRET) between fluorescent proteins fused to the C-termini of LRRC8 subunits. Inter-subunit FRET from LRRC8 complexes tracked VRAC activation. With patch-clamp fluorometry, we confirmed that the cytoplasmic domains rearrange during VRAC opening. With these FRET reporters, we determined VRAC activation, non-invasively, in live cells and their subcompartments. Reduced intracellular ionic strength did not directly activate VRACs, and VRACs were not activated on endomembranes. Instead, pharmacological manipulation of diacylglycerol (DAG), and protein kinase D (PKD) activity, activated or inhibited plasma membrane-localized VRACs. Finally, we resolved previous contradictory reports concerning VRAC activation, using FRET to detect robust activation by PMA that was absent during whole-cell patch clamp. Overall, non-invasive VRAC measurement by FRET is an essential tool for unraveling its activation mechanism.

To determine the value of aerosol amphotericin B inhalations for prevention of invasive pulmonary aspergillosis (IPA), we initiated a prospective randomized multicenter trial. The scheduled intent-to-treat interim analysis included 115 patients (30%) with prolonged neutropenia after chemotherapy for acute myeloid leukemia, acute lymphoblastic leukemia/high-grade non-Hodgkin's lymphoma, or solid tumors undergoing autologous stem cell transplantation. Sixty-five patients had been randomized to receive prophylactic aerosol amphotericin B inhalations at a dose of 10 mg twice daily (group A); for the remaining 50 patients no aerosol amphotericin B prophylaxis was used (group B). No serious side effects from amphotericin B inhalations occurred, but coughing (54%), bad taste (51%), and nausea (37%) caused early cessation of aerosol amphotericin B prophylaxis in 23% (15/65) of courses. In group A, the incidence of proven, probably, or possible IPA was 5% (3/65) as compared with 12% (6/50) in group B (p > 0.05). Microbiologically documented bacterial pneumonias were observed in 5/65 (8%) patients in group A and in 1/50 (2%) patients in group B (p > 0.05). Thus, no reduction in incidence of IPA from use of prophylactic aerosol amphotericin B inhalations was found in this interim analysis. As there were no serious side effects from aerosol amphotericin B prophylaxis, accrual in the study will continue for a total of 380 patients.

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