Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane μH chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igαβ. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and λ5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igαβ in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of λ5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of λ5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.

Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X(3) receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X(3) receptors. At a negative holding potential, the P2X(3) receptor agonist alpha,beta-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba(2+)-containing superfusion medium, or when the buffering of intrapipette Ca(2+) was altered. The C-terminal truncation at Glu362 of P2X(3) receptors abolished the TRPV1/P2X(3) cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X(3) showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X(3) interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X(3) receptor distal to Glu362.

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X 3 receptors. In HEK293 cells permanently transfected with human (h) P2X 3 receptors (HEK293-hP2X 3 cells), an acidic pH shifted the concentration-response curve for ␣,␤-methylene ATP (␣,␤-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of ␣,␤-meATP. Further, a low pH value increased the activation time constant ( on ) of the ␣,␤-meATP current; the fast and slow time constants of desensitization ( des1 , des2 ) were at the same time also increased. Finally, acidification accelerated the recovery of P2X 3 receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X 3 receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the ␣,␤-meATP currents. The voltage sensitivity and reversal potential of the currents activated by ␣,␤-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X 3 receptor channels. It is suggested that acidification may have a dual effect on P2X 3 channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues.High proton concentrations have been registered in inflamed tissue (down to pH 5.4), after surgical interventions (down to pH 5.5), in fracture-related hematomas (down to pH 4.7), in cardiac ischemia (down to pH 5.7), and in and around malignant tumors (1-5). Therefore, local acidosis is considered to contribute to pain experienced in these states (5-11). It is also known that continuous administration of low pH buffered solutions into human skin evokes instant pain and hyperalgesia to mechanical stimulation (12). Electrophysiological experiments in rat skin nerve preparations showed that pathophysiologically relevant high proton concentrations produce a selective nonadapting excitation of nociceptors and a significant sensitization to mechanical stimulation (13). Thus, it has been proposed that local acidosis may play a major role in pain and hyperalgesia (7).Hydrogen ions are able to excite dorsal root ganglion (DRG) 2 neurons via the activation and/or modulation of inward cationselective currents, including the acid-sensing ion channels (ASICs) (14), the transient receptor potential vanilloid receptor 1 (TRPV1) (15, 16), and P2X receptors (17, 18). P2X receptors represent a family of ligand-gated cationic channels that open in response to the binding of ATP, possess two transmembrane domains, intracellular N and C termini, a large extracellular loop, and assemble as hom...

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