The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL − , mscS − , mscK −) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose–response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive “dashpot.” With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of “dumping” a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.

Many cortical network models use recurrent coupling strong enough to require inhibition for stabilization. Yet it has been experimentally unclear whether inhibition-stabilized network (ISN) models describe cortical function well across areas and states. Here, we test several ISN predictions, including the counterintuitive (paradoxical) suppression of inhibitory firing in response to optogenetic inhibitory stimulation. We find clear evidence for ISN operation in mouse visual, somatosensory, and motor cortex. Simple two-population ISN models describe the data well and let us quantify coupling strength. Although some models predict a non-ISN to ISN transition with increasingly strong sensory stimuli, we find ISN effects without sensory stimulation and even during light anesthesia. Additionally, average paradoxical effects result only with transgenic, not viral, opsin expression in parvalbumin (PV)-positive neurons; theory and expression data show this is consistent with ISN operation. Taken together, these results show strong coupling and inhibition stabilization are common features of the cortex.

We describe a mechanism connecting the adaptive behavior of the bacterial mechanosensitive channel MscS to the flexibility of the pore-lining helix TM3. Simulated expansion of the channel structure revealed straightening of a characteristic kink near Gly113 in the open state; return to the closed state produced an alternative kink at Gly121. Patch-clamp experiments showed that higher helical propensity introduced by a G113A mutation prevented inactivation. A similar mutation, G121A, kinetically impeded both closure and inactivation. Duplicating the glycines at each of these sites to increase flexibility produced directly opposite effects. The severely toxic G113A G121A mutation resulted in channels that could not inactivate or close with the release of tension. These data suggest that the open MscS features straight TM3 helices, which act as collapsible 'struts'. Closure and desensitization rely on buckling at Gly121, whereas the crystal-like kink at Gly113 is a feature of the inactivated state.

Summary Plants produce naturally occurring insect repellents, such as citronellal, which is the main component of citronellal oil and is among the most widely-used-naturally-occurring insect repellents. However, the molecular pathways through which insects sense botanical repellents are unknown. Here, we showed that Drosophila used two pathways for direct avoidance of citronellal. The olfactory co-receptor, Or83b, which is required for the response to the synthetic repellent DEET, contributed to citronellal repulsion, and was essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel, TRPA1 resulted in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relied on a G protein/phospholipase C (PLC) signaling cascade, rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq or PLC caused an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel, Slowpoke, resulted in a similar impairment in citronellal avoidance, and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials, and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 was directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria.