The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon's protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.

1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time- and concentration-dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non-responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first-order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist-mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist-mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM-carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM-SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half-maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6-fold in the presence of 0.3 microM-SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist-mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist-mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of ryanodine receptors on the sarcoplasmic reticulum of single smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+]i), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2+]i, one needs to separately monitor Ca2+ entry through them. Such information was obtained from simultaneous measurements of ionic currents and [Ca2+]i by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that approximately 20% of the current passing through the membrane channels activated following caffeine application is carried by Ca2+. Based on this information we found that while Ca2+ entry through these channels rises slowly, release of Ca2+ from stores, while starting at the same time, is much faster and briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was markedly diminished following the second caffeine application whereas the current carried in part by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between ryanodine receptors on the sarcoplasmic reticulum and the nonselective cation channels on the surface membrane.

1. The desensitization of nicotinic acetylcholine receptors on the PC12 sympathetic cell line was investigated by using a 22Na+ influx assay to measure receptor activation. 2. The rate of desensitization was dependent on temperature and at 4 degrees C two distinct kinetic phases were readily discernible: a rapid phase that was characterized by rate constants that were dependent on the chemical nature and concentration of the agonist, and a slower phase that was characterized by rate constants that were less dependent on these. 3. For acetylcholine, carbamylcholine and l-nicotine, the equilibrium desensitization parameter, Kdes, the concentration that produces half-maximal desensitization, was determined and compared with the corresponding value for Kact, the concentration that results in a half-maximal increase in the permeability response. For each agonist, the value of Kdes was found to be lower than Kact, a result to be expected if desensitization is associated with a higher-affinity state of the receptor than that associated with ion channel activation. Thus, extensive receptor desensitization can occur even at agonist concentrations that do not produce appreciable channel activation. Both activation and desensitization functions exhibited positive cooperativity so that each function occurs over a narrow range of agonist concentrations. 4. Following removal of the agonist, recovery from desensitization was reversible and occurred by two distinct kinetic phases characterized by rate constants that were independent of the chemical nature and concentration of the agonist that produced the desensitization. The relative contribution of each kinetic phase of recovery was, however, dependent on the duration of prior exposure to agonist. Following short incubation periods with agonist, most of the receptors were in a rapidly recovering state. With increasing duration of exposure, progressively more of the receptors were converted to a desensitized state that recovered more slowly. 5. The rate constants associated with the two kinetic phases of recovery were dependent on the recovery temperature. Following the initial rapid phase of desensitization, recovery at 4 degrees C was characterized by a time constant, t1/2, of 1.9 min, a value that was about 3-fold greater than that observed at 22 degrees C. The rate of recovery of the desensitized state achieved following equilibrium exposures to agonists was considerably more temperature dependent: recovery of this desensitized state was characterized at 4 degrees C by a t1/2 of 62 min that was about 37-fold greater than that at 22 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)