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.

Putative mechanisms of induction and maintenance of seizure-like activity (SLA) in the low Mg(2+) model of seizures are: facilitation of NMDA receptors and decreased surface charge screening near voltage-gated channels. We have estimated the role of such screening in the early stages of SLA development at both physiological and room temperatures. External Ca(2+) and Mg(2+) promote a depolarization shift of the sodium channel voltage sensitivity; when examined in hippocampal pyramidal neurons, the effect of Ca(2+) was 1.4 times stronger than of Mg(2+). Removing Mg(2+) from the extracellular solution containing 2 mM Ca(2+) induced recurrent SLA in hippocampal CA1 pyramidal layer in 67% of slices. Reduction of [Ca(2+)](o) to 1 mM resulted in 100% appearance of recurrent SLA or continuous SLA. Both delay before seizure activity and the inter-SLA time were significantly reduced. Characteristics of seizures evoked in low Mg(2+)/1 mM Ca(2+)/3.5 K(+) were similar to those obtained in low Mg(2+)/2 Ca(2+)/5mM K(+), suggesting that reduction of [Ca(2+)](o) to 1 mM is identical to the increase in [K(+)](o) to 5 mM in terms of changes in cellular excitability and seizure threshold. An increase of [Ca(2+)](o) to 3 mM completely abolished SLA generation even in the presence of 5 mM [K(+)](o). A large variation in the ability of [Ca(2+)](o) to stop epileptic discharges in initial stage of SLA was found. Our results indicate that surface charge of the neuronal membrane plays a crucial role in the initiation of low Mg(2+)-induced seizures. Furthermore, our study suggests that Ca(2+) and Mg(2+), through screening of surface charge, have important anti-seizure and antiepileptic properties.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

It is well established that temperature affects the functioning of almost all biomolecules and, consequently, all cellular functions. Here, we show how temperature variations within a physiological range affect primary afferents’ spontaneous activity in response to chemical nociceptive stimulation. An ex vivo mouse hind limb skin-saphenous nerve preparation was used to study the temperature dependence of single C-mechanoheat (C-MH) fibers’ spontaneous activity. Nociceptive fibers showed a basal spike frequency of 0.097 ± 0.013 Hz in control conditions (30°C). Non-surprisingly, this activity decreased at 20°C and increased at 40°C, showing moderate temperature dependence with Q10∼2.01. The fibers’ conduction velocity was also temperature-dependent, with an apparent Q10 of 1.38. Both Q10 for spike frequency and conduction velocity were found to be in good correspondence with an apparent Q10 for ion channels gating. Then we examined the temperature dependence of nociceptor responses to high K+, ATP, and H+. Receptive fields of nociceptors were superfused with solutions containing 10.8 mM K+, 200 μM ATP, and H+ (pH 6.7) at three different temperatures: 20, 30, and 40°C. We found that at 30 and 20°C, all the examined fibers were sensitive to K+, but not to ATP or H+. At 20°C, only 53% of fibers were responsible for ATP; increasing the temperature to 40°C resulted in 100% of sensitive fibers. Moreover, at 20°C, all observed fibers were silent to pH, but at 40°C, this number was gradually increased to 87.9%. We have found that the temperature increase from 20 to 30°C significantly facilitated responses to ATP (Q10∼3.11) and H+ (Q10∼3.25), leaving high K+ virtually untouched (Q10∼1.88 vs. 2.01 in control conditions). These data suggest a possible role of P2X receptors in coding the intensity of non-noxious thermal stimuli.