Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na+-selective I1 state to the I2 state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X2 (rP2X2) channels does not occur at mouse P2X2 (mP2X2) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X2 and mP2X2 channels have a pore diameter of ∼11 Å in the I1 state, but the transition to the I2 state increases the rP2X2 diameter by at least 3 Å. The I1 to I2 transition occurs with a rate constant of ∼0.5 s−1. The data focus attention on specific residues of P2X2 channel cytoplasmic domains as determinants of permeation in a state-specific manner.

Nested-primer polymerase chain reaction (PCR) has been applied to the molecular cloning of 4.6-kb half-genome fragments of human immunodeficiency virus type 1 (HIV-1) taken directly from the peripheral blood mononuclear cells (PBMC) of an individual with neurological symptoms of HIV-1 infection. In a similar manner, gpl20-coding portions of the envelope gene were cloned after PBMC from the same blood sample were cocultivated with uninfected PBMC for 28 days. The complete 1.6-kb nucleotide sequence of the gpl20 gene was determined from each of 35 clones examined. Two of 13 (15%) PBMC-derived gpl20 genes and 3 of 22 (14%) coculture-derived gpl20 genes were defective as a result of frameshifts and an in-frame stop codon(s). Mean diversity between individual gpl20-coding sequences in PBMC was fivefold greater (3.24%) than after coculture (0.65%). A predominant sequence or "strain" was found after coculture that was distinct from the diverse viral genotypes detected in vivo and therefore was selectively amplified during in vitro propagation. Multiple distinct third variable (V3) regions encoding the principal neutralizing domain of the envelope protein were detected in PBMC-derived genes, suggesting the presence of immunologic diversity of HIV env genes in vivo not reflected in the cocultured virus sample. The large size of the HIV fragments generated in this study will permit analysis of the diversity of immunologic reactivity, gene function, and pathogenicity of HIV genomes present within infected individuals, including the functional significance of the loss of diversity that occurs upon coculture. * Corresponding author. t We wish to acknowledge the memory of our dear colleague Donald Vaughn Faulkner. Don launched and nurtured many of us into the computer age, and he is greatly missed.

PTEN/MMAC1 is a recently characterized tumor suppressor. A pseudogene derived from the human PTEN/MMAC1 phosphatase, CPTEN, has been reported. Recent analysis of the pseudogene revealed con¯icting results about the expression of CPTEN. In this study, we show that the PTEN/MMAC1 pseudogene is actively transcribed in all cells and tissues examined. In some cases, pseudogene transcripts were found to represent as much as 70% of the total PTEN/MMAC1 RNA. As CPTEN is transcribed, there is a potential for misinterpretation of PTEN/MMAC1 mutations when RT ± PCR techniques are used, as well as potential for a CPTEN-encoded translation product. Although we were unable to detect a pseudogene protein product in the cell lines examined, a baculovirus produced GST pseudogene fusion protein exhibited phosphatase activity comparable to wild type. The results of this study, taken together, indicate the potential complication of PTEN/MMAC1 molecular analysis caused by the expression of CPTEN.

Bruton's tyrosine kinase (Btk) 1 is a member of the Tec family of nonreceptor protein-tyrosine kinases (PTKs) that includes Bmx, Itk, Tec, and Txk (reviewed in Ref. 1). Tec family PTKs are characterized by an NH 2 -terminal pleckstrin homology (PH) domain followed by a Tec homology domain (containing a Zn 2ϩ -binding Btk motif and a proline-rich region), a Src-homology 3 (SH3) and SH2 domain, and a COOH-terminal kinase domain (1, 2) (Fig. 1). Tec PTKs share 50 -60% amino acid sequence identity and are predominately expressed by cells of the immune system (1). Bmx and Txk are somewhat atypical family members: Bmx shares little sequence homology with other Tecs within the proline-rich and SH3 domains; Txk is particularly atypical at the NH 2 terminus, lacking the PH and Tec homology domains (Fig.