SummaryThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.

Physiological response to thermal stimuli in mammals is mediated by a structurally diverse class of ion channels, many of which exhibit polymodal behavior. To probe the diversity of biophysical mechanisms of temperature-sensitivity, we characterized the temperature-dependent activation of MthK, a two transmembrane calcium-activated potassium channel from thermophilic archaebacteria. Our functional complementation studies show that these channels are more efficient at rescuing K+ transport at 37°C than at 24°C. Electrophysiological activity of the purified MthK is extremely sensitive (Q10 >100) to heating particularly at low-calcium concentrations whereas channels lacking the calcium-sensing RCK domain are practically insensitive. By analyzing single-channel activities at limiting calcium concentrations, we find that temperature alters the coupling between the cytoplasmic RCK domains and the pore domain. These findings reveal a hitherto unexplored mechanism of temperature-dependent regulation of ion channel gating and shed light on ancient origins of temperature-sensitivity.

In the global trend of urban shrinkage, urban vitality, as one of the important representations of high-quality urban development, has become a breakthrough. More and more scholars advocate to awaken urban vitality, so as to realize the high-quality development of shrinking cities. This paper takes the municipal districts of 34 cities in the three northeastern provinces of China as study areas, based on the broad concept of urban shrinkage, selects the indicators of population, economy and society, and uses the “two-step diagnostic method” which is consistent with Chinese conditions to identify the urban shrinkage from 2010 to 2018. In this research, the indexes of economic, social, cultural, environmental and spatial dimensions are selected, and the urban vitality and the vitality of each dimension from 2010 to 2018 are calculated and analyzed by using the entropy weight method (EWM). Then, this paper analyzes the correlation between urban shrinkage and urban vitality by Pearson correlation coefficient. The results show that: (1) urban shrinkage in the three northeastern provinces of China has become a regional remarkable phenomenon, which is also an inevitable process in some regions of China and even the world; (2) overall, the urban vitality of cities in the three northeastern provinces of China is steady and rising a little, and there is an obvious spatial agglomeration pattern like “central city polarization”; (3) there is a significant correlation between urban shrinkage and urban vitality, that is, the lower the degree of urban shrinkage, the higher the urban vitality, showing the opposite trend in the process of urban development; (4) the influence of urban shrinkage on each dimension of urban vitality is different, and the correlation results are different, too. In the planning process of shrinking cities in the future, paying attention to the relationship between urban vitality and urban shrinkage, conducting benign guidance on this basis, and adjusting urban vitality elements of different dimensions to stimulate urban development power can enhance urban competitiveness and achieve better development.

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