Nonadiabatic bridge-assisted electron transfer ͑ET͒ is described by a set of kinetic equations which simultaneously account for the sequential ͑hopping͒ as well as the superexchange mechanism. The analysis is based on the introduction of a certain reduced density operator describing a particular set of electron-vibrational levels of the molecular units ͑sites͒ involved in the transfer act. For the limiting case of intrasite relaxations proceeding fast compared to intersite transitions a set of rate equations is obtained. This set describes the time evolution of the electronic site populations and is valid for bridges with an arbitrary number of units. If the rate constants for the transition from the bridge to the donor as well as to the acceptor exceed those for the reverse transitions the ET reduces to a single-exponential process with an effective forward and backward transfer rate. These effective rates contain a contribution from the sequential and a contribution from the superexchange mechanisms. A detailed analysis of both mechanisms is given showing their temperature dependence, their dependence on the number of bridge units, and the influence of the energy gap and the driving force. It is demonstrated that for integral bridge populations less than 10 Ϫ3 the complicated bridge-mediated ET reduces to a donor-acceptor ET with an effective overall transfer rate. This transfer rate contains contributions from the sequential as well as the superexchange mechanisms, and thus can be used for a quantitative analysis of the efficiency of different electron pathways. For room-temperature conditions and even at a very small bridge population of 10 Ϫ4-10 Ϫ10 the superexchange mechanism is superimposed by the sequential one if the number of bridge units exceeds 4 or 5.

P2X3 purinoreceptors expressed in mammalian sensory neurons are involved in nociception, mechanosensory transduction, and temperature sensation. Homomeric P2X3 receptors desensitize rapidly (<500 ms after activation by an agonist) and recover from desensitization very slowly (20-25 min at room temperature). They are susceptible to use-dependent inhibition by low nanomolar concentrations of ATP through developing the "high-affinity binding site" (HABS), which traps ATP molecules, thus keeping receptors in a desensitized state (Pratt et al., J Neurosci 25:7359-7365, 2005). Indeed, here we demonstrated directly that the desensitization of the receptor, after being activated by ATP, proceeds independently of the presence of agonist. We found that the temperature sensitivity of P2X3 receptors is abnormal: development of desensitization does not depend on temperature within the range between 25 and 40 degrees C, whereas the recovery from desensitization is greatly \accelerated with temperature increase (Q10 approximately 10). The sensitivity of HABS to low nanomolar ATP near normal body temperature (35 degrees C) is substantially lower than at 25 degrees C (IC50 is 3.2+/-0.3 nM at 35 degrees C and 0.79+/-0.09 nM at 25 degrees C). HABS itself is subjected to slow desensitization partially loosing its sensitivity to ATP: at 35 degrees C the response completely recovers in 10 min in the presence of 3 nM ATP, making the receptor operational in the presence of up to 30 nM ATP. Unusual combination of temperature sensitivity/insensitivity of P2X3 receptors may be related to their pivotal role in the processing of thermal sensitivity as revealed by recent knockout experiments.

A master equation describing the evolution of averaged molecular state occupancies in molecular systems where alternation of molecular energy levels is caused by discrete dichotomous and trichotomous stochastic fields, is derived. This study is focused on the kinetics of quasi-isoenergetic transition processes in the presence of moderately high frequency stochastic field. A novel physical mechanism for temperature-independent transitions in flexible molecular systems is proposed.This mechanism becomes effective when the conformation transitions between quasiisoenergetic molecular states take place. At room temperatures, stochastic broadening of molecular energy levels predominates the energy of low frequency vibrations accompanying the transition. This leads to a cancellation of the temperature dependence in the stochastically averaged rate constants. As examples, physical interpretations of the temperature-independent onset of P2X 3 receptor desensitization in neuronal membranes, as well as degradation of PER2 protein in embrionic fibroblasts, are provided.

SUMMARY1. Single low-threshold inactivating (LTI or T-type) Ca2+ channels of undifferentiated neuroblastoma cells (clone NlE-115) were investigated using the patchclamp technique.2. Single-channel conductance, gi, for Ca2 , Sr2+ or Ba2+ as a permeant cation was similar (7-2 pS). Mean channel open time, r0O, was also practically independent of the divalent ion species; it decreased from 0-7 to 0-3 ms between -40 and 0 mV.3. Modification of the calcium channel selectivity by lowering the external Ca2+ concentration to 10-8 M produced an increase in gi for Na+ and Li+ ions and a shift of potential-dependent characteristics in the hyperpolarizing direction. Voltage sensitivity and absolute values of Top were also changed. These changes were dependent on both permeant monovalent ion type and concentration.4. At high [Na+]0, T0p was almost potential independent (-0 3 ms). Decrease in [Na+]. and substitution of Li+ for Na+ increased T0p and the steepness of its potential dependency.5. The divalent and monovalent cations that were tested had much smaller effect on the mean intraburst shut time, Tel(f), which was nearly independent of membrane potential (-0-6 ms). By contrast, mean burst duration was strongly potential dependent and noticeably affected by permeant ion type.6. All kinetic changes were analysed in terms of a four-state sequential model for channel activation. According to this model the channel enters the open state through three closed states. Transitions between closed states can be formally related to the transmembrane movement of two charged gating particles (M2 process). The interaction between ion flux and a sterical region of the Ca2+ channel selectivity filter may, depending on ion transfer rate and ionic radius, lead to a local increase of the dielectric constant, resulting in redistribution of the electric field and changes in potential dependency of r.p. MS 8874 YA. M. SHUBA AND OTHERS

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