The Israel Society for Neuroscience (ISFN) was founded in 1993 by a group of Israeli leading scientists conducting research in the area of neurobiology. The primary goal of the society was to promote and disseminate the knowledge and understanding acquired by its members, and to strengthen interactions between them. Since then, the society holds its annual meeting every year in Eilat during the month of December. At these annual meetings the senior Israeli neurobiologists, their teams, and their graduate students, as well as foreign scientists and students, present their recent research findings in platform and poster presentations. The meeting also offers the opportunity for the researchers to exchange information with each other, often leading to the initiation of collaborative studies. Both the number of members of the society and of those participating in the annual meeting is constantly increasing, and it is anticipated that this year about 600 scientists will convene at the Princess Hotel in Eilat, Israel. Further information concerning the Israel Society for Neuroscience can be found at http://www.isfn.org.il. Committee: Zvi Wollberg (President) Tel Aviv University Edi Barkai University of Haifa Etti Grauer Israel Institute for Biological Research, Ness Ziona Yoram Rami Grossman Ben Gurion University of the Negev Yoel Yaari Hebrew University of Jerusalem Gal Yadid Bar-Ilan University Shlomo Rotshenker (President Elect) Hebrew University of Jerusalem Ettie Grauer (Treasurer) Israel Institute for Biological Research, Ness Ziona Michal Gilady (Administrator) Rishon Le Zion

In voltage-dependent ether-à-go-go (eag) K + channels, the process of activation is modulated by Mg 2+ and other divalent cations, which bind to a site in the voltage sensor and slow channel opening. Previous analysis of eag ionic and gating currents indicated that Mg 2+ has a much larger effect on ionic than gating current kinetics. From this, we hypothesized that ion binding modulates voltage sensor conformational changes that are poorly represented in gating current recordings. We have now tested this proposal by using a combined electrophysiological and optical approach. We find that a fluorescent probe attached near S4 in the voltage sensor reports on two phases of the activation process. One component of the optical signal corresponds to the main charge-moving conformational changes of the voltage sensor. This is the phase of activation that is well represented in gating current recordings. Another component of the optical signal reflects voltage sensor conformational changes that occur at more hyperpolarized potentials. These transitions, which are rate-determining for activation and highly modulated by Mg 2+ , have not been detected in gating current recordings. Our results demonstrate that the eag voltage sensor undergoes conformational changes that have gone undetected in electrical measurements. These transitions account for the time course of eag activation in the presence and absence of extracellular Mg 2+ .

Our central nervous system is based on the generation and propagation of electrical signals along the neuronal pathways. These variations of the membrane potential are arranged by the concerted action of ion channels in the neuronal membrane. Therefore, the exact measurement of the electric field in the central nervous system is the focus of intensive investigation. While electrophysiological methods provide exact measurements on the single-cell or single-molecule level with high temporal resolution, they are limited in their spatial resolution ranging from a few single cells to a single molecule. To thoroughly understand how the voltage-dependent ion channels sense the membrane potential and are precisely gated by it, the electric field within the protein has to be investigated. Likewise, the propagation of electrical impulses in a network of neurons involves a large number of cells, which have to be monitored simultaneously. For these endeavors, optical methods have proven to be useful due to their scalability, temporal and spatial resolution. Here, we will summarize the properties of the optical probes that we used to determine the electrical field strength within voltage-sensitive ion channels and discuss the hybrid approach to detect membrane potential changes in genetically specified neurons in terms of design, limitations and future developments.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the β19′ position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Δ F / F ) ≈10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose–response relations for Δ F agreed well with those for epibatidine-induced currents, but were shifted ≈100-fold to the left of those for ACh-induced currents. Because ( i ) epibatidine binds more tightly to the αγ-binding site than to the αδ site and ( ii ) ACh binds with reverse-site selectivity, these data suggest that Δ F monitors an event linked to binding specifically at the αδ-subunit interface. In experiments with flash-applied agonists, the earliest detectable Δ F occurs within milliseconds, i.e., during activation. At low [ACh] (≤ 10 μM), a phase of Δ F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Δ F is complete before the slowest phase of recovery from desensitization (time constant ≈250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the αδ-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains (Chanda, B., and F. Bezanilla. 2002. J. Gen. Physiol. 120:629–645). Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.