Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

The insulin-like signaling pathway is known to regulate fat metabolism, dauer formation, and longevity in Caenorhabditis elegans. Here, we report that this pathway is also involved in salt chemotaxis learning, in which animals previously exposed to a chemoattractive salt under starvation conditions start to show salt avoidance behavior. Mutants of ins-1, daf-2, age-1, pdk-1, and akt-1, which encode the homologs of insulin, insulin/IGF-I receptor, PI 3-kinase, phosphoinositide-dependent kinase, and Akt/PKB, respectively, show severe defects in salt chemotaxis learning. daf-2 and age-1 act in the ASER salt-sensing neuron, and the activity level of the DAF-2/AGE-1 pathway in this neuron determines the extent and orientation of salt chemotaxis. On the other hand, ins-1 acts in AIA interneurons, which receive direct synaptic inputs from sensory neurons and also send synaptic outputs to ASER. These results suggest that INS-1 secreted from AIA interneurons provides feedback to ASER to generate plasticity of chemotaxis.

It is poorly understood how sensory systems memorize the intensity of sensory stimulus, compare it with a newly sensed stimulus, and regulate the orientation behaviour based on the memory. Here we report that Caenorhabditis elegans memorizes the environmental salt concentration during cultivation and exhibits a strong behavioural preference for this concentration. The right-sided amphid gustatory neuron known as ASER, senses decreases in salt concentration, and this information is transmitted to the postsynaptic AIB interneurons only in the salt concentration range lower than the cultivation concentration. In this range, animals migrate towards higher concentration by promoting turning behaviour upon decreases in salt concentration. These observations provide a mechanism for adjusting the orientation behaviour based on the memory of sensory stimulus using a simple neural circuit. A ll living animals are endowed with the capacity to seek optimal ambient conditions. Such behaviour requires memorizing the intensity of sensory stimulus associated with favourable conditions, recognizing the spatio-temporal changes of the stimulus intensity under current conditions, comparing it with the memorized stimulus intensity and regulating the motor output to generate a movement towards a preferred direction. However, in most sensory systems how stimulus intensity is memorized and how distinct orientation behaviours are generated depending on the memory have been poorly explored.Sodium chloride was identified as a chemoattractant for C. elegans 1 . Based on this view the mechanisms of salt chemotaxis has been examined for decades. Amphid taste neurons ASE, which consist of two morphologically symmetric neurons on the left (ASEL) and the right (ASER), have a predominant role in chemotaxis to NaCl and other inorganic salts 2-4 . Calcium imaging experiments showed that these neurons are functionally asymmetric: ASER is depolarized by decreases in salt concentration, while ASEL responds to increases 5 . As activation of ASER and ASEL promotes redirecting turns and forward locomotion, respectively, it was suggested that the right and the left neurons have distinct roles but act in concert for migrating up salt gradient 5 . Through another line of studies on the migration behaviours, two distinct behavioural mechanisms that drive animals towards higher salt concentration have been characterized so far (see below) 6,7 . Despite these sets of knowledge, the physiological and mechanistic properties of the information flow for salt chemotaxis downstream of the sensory neurons have not been obvious, as ASEL and ASER form synapses onto overlapping targets (Fig. 1a).Meanwhile, we have previously shown that pairing starvation with exposure to NaCl causes salt avoidance learning in C. elegans 8 . The insulin signalling pathway components, DAF-2 insulin receptor, AGE-1 phosphoinositide 3-kinase and AKT-1 AKT kinase, are required in ASER for this learning 9 . Activation of the Gq/DAG/PKC signalling pathway consisting of EGL-30 Gq, diacylglycer...

The phosphatidylinositol 3-kinase (PI3K) pathway regulates many cellular functions, but its roles in the nervous system are still poorly understood. We found that a newly discovered insulin receptor isoform, DAF-2c, is translocated from the cell body to the synaptic region of the chemosensory neuron in Caenorhabditis elegans by a conditioning stimulus that induces taste avoidance learning. This translocation is essential for learning and is dependent on the mitogen-activated protein kinase-regulated interaction of CASY-1 (the calsyntenin ortholog) and kinesin-1. The PI3K pathway is required downstream of the receptor. Light-regulated activation of PI3K in the synaptic region, but not in other parts of the cell, switched taste-attractive behavior to taste avoidance, mimicking the effect of conditioning. Thus, synaptic PI3K is crucial for the behavioral switch caused by learning.