Conductance stability and Na+ interaction with Shab K+ channels under low K+ conditions.

K+ ions exert a structural effect that brings stability to K+ selective pores. Thus, upon bathing Shab channels in 0 K+ solutions the ion conductance, GK, irreversibly collapses. Related to this, studies with isolated KcsA channels have suggested that there is a transition [K+] around which the pore takes one of two conformations, either the low (non-conducting) or high K+ (conducting) crystal structures. We examined this premise by looking at the K+-dependency of GK stability of Shab channels within the cell membrane environment. We found that: K+ effect on GK stability is highly asymmetrical, and that as internal K+ is replaced by Na+ GK drops in a way that suggests a transition internal [K+]. Additionally, we found that external permeant ions inhibit GK drop with a potency that differs from the global selectivity-sequence of K+ pores; the non-permeant TEA inhibited GK drop in a K+-dependent manner. Upon lowering internal [K+] we observed an influx of Na+ at negative potentials. Na+ influx was halted by physiological external [K+], which also restored GK stability. Hyperpolarized potentials afforded GK stability but, as expected, do not restore GK selectivity. For completeness, Na+ interaction with Shab was also assessed at depolarized potentials by looking at Na block followed by permeation (pore unblock) at positive potentials, in solutions approaching the 0 K+ limit. The stabilizing effect of negative potentials along with the non-parallel variation of Na+ permeability and conductance-stability herein reported, show that pore stability and selectivity, although related, are not strictly coupled.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Desensitization and recovery of crayfish photoreceptors. Dependency on circadian time, and pigment-dispersing hormone.

In this work, we studied the characteristics of recovery from desensitization of the light-elicited current of crayfish. Applying a two-flash protocol, we found that the first flash triggers a current that activates with a noticeable latency, reaches a peak value, and thereafter decays along a single exponential time course. In comparison with the first-elicited current, the current elicited by the second flash not only presents an expected smaller peak current, depending on the time between flashes, but it also displays a different latency and decay time constant. Recovery of the first flash values of these current parameters depends on the circadian time at which the experiments are conducted, and on the presence of pigment-dispersing hormone. Our data also suggest the existence of distinctive desensitized states, whose induction depends on circadian time and the presence of pigment-dispersing hormone.

Modeling some properties of circadian rhythms.

Mathematical models have been very useful in biological research. From the interaction of biology and mathematics, new problems have emerged that have generated advances in the theory, suggested further experimental work and motivated plausible conjectures. From our perspective, it is absolutely necessary to incorporate modeling tools in the study of circadian rhythms and that without a solid mathematical framework a real understanding of them will not be possible. Our interest is to study the main process underlying the synchronization in the pacemaker of a circadian system: these mechanisms should be conserved in all living beings. Indeed, from an evolutionary perspective, it seems reasonable to assume that either they have a common origin or that they emerge from similar selection circumstances. We propose a general framework to understand the emergence of synchronization as a robust characteristic of some cooperative systems of non-linear coupled oscillators. In a first approximation to the problem we vary the topology of the network and the strength of the interactions among oscillators. In order to study the emergent dynamics, we carried out some numerical computations. The results are consistent with experiments reported in the literature. Finally, we proposed a theoretical framework to study the phenomenon of synchronization in the context of circadian rhythms: the dissipative synchronization of nonautonomous dynamical systems.

Induction of a fast inactivation gating on delayed rectifier Shab K(+) channels by the anti-inflammatory drug celecoxib.

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K(+) channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of I(K). Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.

Effect of pigment dispersing hormone on the electrical activity of crayfish visual photoreceptors during the 24-h cycle.

Visual photoreceptors are structures involved in the expression and synchronization of crayfish circadian rhythm of sensitivity to light (electroretinogram, ERG). Considering the relevant role of Pigment dispersing hormone (PDH) in the invertebrate circadian system organization, we study the effect of this substance on the electrical activity of crayfish visual photoreceptors during the 24-h cycle. The study demonstrates that: (1) PDH affects the electrical response to light of crayfish visual photoreceptor cells in a circadian time-dependent manner. (2) The kinetics of the light-elicited current of crayfish visual photoreceptor cells, as well as the ionic permeability underlying the electrical response to light vary over the 24-h cycle. (3) PDH modifies the kinetics and ionic permeability underlying the light-elicited current of crayfish visual photoreceptor cells in a circadian time-dependent manner.