We have characterized the maturation of Shaker K ؉ channel protein and the cellular site of assembly of pore-forming ␣ and cytoplasmic ␤ subunits in a transfected mammalian cell line. Shaker protein is made as a partially glycosylated, immature precursor that is converted to a fully glycosylated, mature product. Shaker protein did not mature when transport from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Consistent with this finding, only the immature form was sensitive to digestion with endoglycosidase H. These results indicate that the immature protein is coreglycosylated in the ER, whereas the oligosaccharides of the mature protein have been further processed in the Golgi compartment. After inhibiting ER-to-Golgi transport, the oligomeric state of Shaker subunits was assessed by cross-linking in intact cells or by solubilization and sucrose gradient sedimentation. The results indicate that Shaker subunits assemble with each other in the ER. When co-expressed, the Kv␤2 subunit also associated with Shaker in the ER. Assembly with the ␤2 subunit did not increase the rate or extent of Shaker protein maturation. Our results indicate that the biogenesis of Shaker K ؉ channels in vivo involves core glycosylation and subunit assembly in the ER, followed by efficient transfer to the Golgi apparatus where the oligosaccharides are modified.

P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X 2 receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X 2 receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His 3 Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His 120 and His 213 sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X 2 receptors.P2X receptors are oligomeric, ATP-gated cation channels that are distributed widely throughout the central and peripheral nervous systems of mammals and are known to be involved in fast excitatory synaptic transmission (1). P2X receptors are structurally distinct from the nicotinic receptor and ionotropic glutamate receptor channel superfamilies. In mammals, the P2X family has seven members encoded by different genes (P2X 1-7 ) that can associate as homo-and heteromeric channel assemblies (1). P2X receptors are thought to be trimers (2) with subunits that have intracellular N and C termini, two transmembrane domains, and a large extracellular loop (1).Colocalization of zinc with P2X 2 receptors in the nervous system suggests a physiological role for this divalent cation in modulating ATP-evoked currents (3, 4). Extracellular zinc potentiates P2X receptor currents in rat sensory and sympathetic neurons as well as in rat PC12 cells (1). The potentiating effect of zinc on P2X 2 receptors results from a decrease in the EC 50 for ATP without a concomitant change in the maximum response to ATP (5, 6).We have previously identified two molecular determinants of zinc potentiation for the rat P2X 2 subunit (6); mutation of either His 120 or His 213 to alanine eliminated potentiation by zinc. Direct participation of these residues in zinc binding is consistent with t...

In the voltage-dependent Shaker K ؉ channel, distinct regions of the protein form the voltage sensor, contribute to the permeation pathway, and recognize compatible subunits for assembly. To investigate channel biogenesis, we disrupted the formation of these discrete functional domains with mutations, including an aminoterminal deletion, ⌬97-196, which is likely to disrupt subunit oligomerization; D316K and K374E, which prevent proper folding of the voltage sensor; and E418K and C462K, which are likely to disrupt pore formation. We determined whether these mutant subunits undergo three previously identified assembly events as follows: 1) tetramerization of Shaker subunits, 2) assembly of Shaker (␣) and cytoplasmic ␤ subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. ⌬97-196 subunits failed to establish any of these quaternary interactions. The ⌬97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the ␤ subunit but did not establish proximity between the amino and carboxyl termini of adjacent subunits. The results indicate that oligomerization mediated by the amino terminus is required for subsequent pore formation and either precedes or is independent of folding of the voltage sensor. In contrast, the amino and carboxyl termini of adjacent subunits associate late during biogenesis. Because subunits with folding defects oligomerize, we conclude that Shaker channels need not assemble from pre-folded monomers. Furthermore, association with native subunits can weakly promote the proper folding of some mutant subunits, suggesting that steps of folding and assembly alternate during channel biogenesis.

Shaker potassium (K+) channels normally lack intrasubunit and intersubunit disulfide bonds. However, disulfide bonds are formed between Shaker subunits in intact cells exposed to oxidizing conditions. Upon electrophoresis under nonreducing conditions, intersubunit disulfide bond formation was detected by the presence of four high molecular weight adducts of Shaker protein. This result suggests that intracellular cysteine residues are in sufficiently close proximity in the native structure of the Shaker channel to form intersubunit disulfide bonds. To test this hypothesis, wild-type and mutant Shaker proteins were exposed to oxidizing conditions in intact cells. Intersubunit disulfide bond formation was eliminated upon serine substitution of either C96 in the amino terminal or C505 in the carboxyl terminal of the protein. In contrast, disulfide bond formation was not eliminated upon serine substitution of both C301 and C308 in the cytoplasmic loop between transmembrane segments S2 and S3. Exposure of Shaker-expressing cells to oxidizing conditions did not significantly alter the amplitude, kinetics, or voltage dependence of the Shaker current, demonstrating that the native tertiary and quaternary structures of the channel were maintained under oxidizing conditions. These results indicate that intersubunit disulfide bonds form between C96 and C505, providing evidence that the amino- and carboxyl-terminal regions of adjacent subunits are in proximity in the native structure of the channel. The disulfide-bonded adducts were found to represent a dimer, a trimer, and two forms of tetramer, one linear and one circular, containing one, two, three, or four disulfide bonds, respectively. These results provide a direct biochemical demonstration that Shaker K+ channels contain four pore-forming subunits.

Trauma- and stress-related disorders are among the most common types of mental illness affecting the U.S. population. For many of these disorders, there is a striking sex difference in lifetime prevalence; for instance, women are twice as likely as men to be affected by posttraumatic stress disorder (PTSD). Gonadal steroids and their metabolites have been implicated in sex differences in fear and anxiety. One example, allopregnanolone (ALLO), is a neuroactive metabolite of progesterone that allosterically enhances GABAA receptor activity and has anxiolytic effects. Like other ovarian hormones, it not only occurs at different levels in males and females but also fluctuates over the female reproductive cycle. One brain structure that may be involved in neuroactive steroid regulation of fear and anxiety is the bed nucleus of the stria terminalis (BNST). To explore this question, we examined the consequences of augmenting or reducing ALLO activity in the BNST on the expression of Pavlovian fear conditioning in rats. In Experiment 1, intra-BNST infusions of ALLO in male rats suppressed freezing behavior (a fear response) to the conditioned context, but did not influence freezing to a discrete tone conditioned stimulus (CS). In Experiment 2, intra-BNST infusion of either finasteride (FIN), an inhibitor of ALLO synthesis, or 17-phenyl-(3α,5α)-androst-16-en-3-ol, an ALLO antagonist, in female rats enhanced contextual freezing; neither treatment affected freezing to the tone CS. These findings support a role for ALLO in modulating contextual fear via the BNST and suggest that sex differences in fear and anxiety could arise from differential steroid regulation of BNST function. The susceptibility of women to disorders such as PTSD may be linked to cyclic declines in neuroactive steroid activity within fear circuitry.