The mechanism of inward rectification in Kir channels: A novel kinetic model with non-equilibrium thermodynamics approach.

The mechanisms of the strong inward rectification in inward rectifier K(+) (Kir) channels are controversial because the drop in electrical potential due to the movement of the blocker and coupling ions is insufficient to explain the steep voltage-dependent block near the equilibrium potential. Here, we study the "driving force"-dependent block in Kir channels with a novel approach incorporating concepts from the non-equilibrium thermodynamics of small systems, and computer kinetic simulations based on the experimental data of internal Ba(2+) block on Kir2.1 channels. The steep exponential increase in the apparent binding rate near the equilibrium potential is explained, when the encounter frequency is construed as the likelihood of transfer events down or against the electrochemical potential gradient. The exponent of flux ratio, nf=2.62, implies that the blockage of the internal blocker may be coupled with the outward transport of 2 to 3K(+) ions. The flux-coupled block in the single-file multi-ion pore can be demonstrated by the concentration gradient alone, as well as when the driving force is the electrochemical potential difference across the membrane.

Driving force-dependent block by internal Ba(2+) on the Kir2.1 channel: Mechanistic insight into inward rectification.

The Kir2.1 channel is characterized by strong inward rectification; however, the mechanism of the steep voltage dependence near the equilibrium potential remains to be investigated. Here, we studied the internal Ba(2+) block of the Kir2.1 channel expressed in Xenopus oocytes. We showed that the driving force and thus the K(+) ion flux significantly influenced the apparent affinity of the block by internal Ba(2+). Kinetic analysis revealed that the binding rate shifted with the driving force and changed steeply near the equilibrium point, either in the presence or absence of the transmembrane electrical field. The unbinding rate was determined by the intrinsic affinity of the site. Mutagenesis studies revealed that the high-affinity binding site for Ba(2+) was located near T141 at the internal entrance of the selectivity filter. The steep change of the blocking affinity near the equilibrium potential may result from the flux-coupling effect in the single-file, multi-ion cytoplasmic pore.

Positive attitudes and self-harming behavior of adolescents in a juvenile detention house in Taiwan.

This study aimed to evaluate the less stigmatizing positivity construct screening measurement and its association with recent self-harming behaviors among adolescents. Participants were 193 detained Taiwanese adolescents. Questionnaires consisted of a deliberate self-harm inventory, a positivity construct measurement, a depression scale, data concerning risky health behaviors and demographics. The prevalence rate of recent self-harming behavior among adolescents in the detention house was 43.5%. The logistic model showed that age, gender and level of positivity demonstrated significant odds ratios for self-harm behavior. Results showed that younger age and female gender increased self-harming behavior. In addition, low score on positivity construct screening measurement increased the probability of self-harming behavior. Furthermore, these adolescents also engaged in risky health behaviors and were more depressed. Parental and school awareness for these risky behaviors should be enhanced and appropriate early interventions implemented to prevent negative health outcomes.

Interpretation of the Ussing flux ratio from the fluctuation theorem.

The fluctuation theorem gives a mathematical expression to quantify the probability of observing events violating the second law of thermodynamics in a small system over a short period of time. The theorem predicts the ratio of forward (entropy-producing) runs to the backward (entropy-consuming) runs for a nanometer-sized molecular machine in a nonequilibrium system. However, few experimental verifications of the theorem have been carried out. In this paper, I show that the Ussing flux ratio, the ratio of outward to inward unidirectional ion fluxes across a membrane channel, can be derived from the fluctuation theorem if we consider the ion channel and the contacting solutions as a small nonequilibrium system. The entropy change due to ion electrodiffusion is expressed from the fundamental equation for the entropy change. Thus, the empirical flux ratio equation can be interpreted from the more general fluctuation theorem, and serves as a verification of the theorem.

Redox modulation of A-type K+ currents in pain-sensing dorsal root ganglion neurons.

Redox modulation of fast inactivation has been described in certain cloned A-type voltage-gated K(+) (Kv) channels in expressing systems, but the effects remain to be demonstrated in native neurons. In this study, we examined the effects of cysteine-specific redox agents on the A-type K(+) currents in acutely dissociated small diameter dorsal root ganglion (DRG) neurons from rats. The fast inactivation of most A-type currents was markedly removed or slowed by the oxidizing agents 2,2'-dithio-bis(5-nitropyridine) (DTBNP) and chloramine-T. Dithiothreitol, a reducing agent for the disulfide bond, restored the inactivation. These results demonstrated that native A-type K(+) channels, probably Kv1.4, could switch the roles between inactivating and non-inactivating K(+) channels via redox regulation in pain-sensing DRG neurons. The A-type channels may play a role in adjusting pain sensitivity in response to peripheral redox conditions.

Use-dependent inhibition of the N-methyl-D-aspartate currents by felbamate: a gating modifier with selective binding to the desensitized channels.

Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect may be related to the inhibition of N-methyl-D-aspartate (NMDA) currents, but the exact molecular action remains unclear. Using whole-cell patch-clamp recording in rat hippocampal neurons, we found that submillimolar FBM effectively modifies the gating process of NMDA channels. During a single high-concentration (1 mM) NMDA pulse, FBM significantly inhibits the late sustained current but not the early peak current. However, if the 1 mM NMDA pulse is preceded by a low-concentration (10 microM) NMDA prepulse, then FBM significantly inhibits both the peak and the sustained currents in the 1 mM pulse. In sharp contrast, the NMDA currents elicited by micromolar NMDA are only negligibly inhibited or even enhanced by FBM. These findings indicate that the inhibitory effect of FBM on NMDA currents is stronger with both higher NMDA concentration and longer NMDA exposure, and is thus "use-dependent". FBM also slows recovery of the desensitized NMDA channel, and quantitative analyses of FBM effects on the activation kinetics and the desensitization curve of the NMDA currents further disclose dissociation constants of approximately 200, approximately 110, and approximately 55 microM for FBM binding to the resting, activated, and desensitized NMDA channels, respectively. We conclude that therapeutic concentrations (50-300 microM) of FBM could bind to and modify a significant proportion of the resting NMDA channel even when NMDA or other glutamatergic ligand is not present and then decrease the NMDA currents at subsequent NMDA pulses by stabilization of the desensitized channels. Because the inhibitory effect is apparent only when there is excessive NMDA exposure, FBM may effectively inhibit many seizure discharges but preserve most normal neuronal firings.

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels.

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.