Toxicity of subretinal ribozyme to the proliferating cell nuclear antigen and 5-fluorouracil in rat eyes.

PURPOSE: To investigate the subretinal toxicity profile of the ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU), as well as the highest nontoxic subretinal dose of the mixture of the two agents in rat eyes. METHODS: Brown-Norway rats received subretinal injections of 1 microg, 10 microg, and 100 microg/microl PCNA-Rz and 0.06 microg/microl, 0.3 microg/microl, and 1.5 microg/microl 5-FU in the right eyes, and the left eyes were injected with H-BSS as control. Each dose was tested on 5 eyes in a 5 microl volume. In a second study, a combination of 5-FU (1.5 microg/microL) with varying 10-30-50 microg/microl doses of PCNA-Rz was tested in a regimen of four sequential subretinal injections. Toxicity was monitored by biomicroscopy, indirect ophthalmoscopy, electroretinography (ERG), and histology. RESULTS: The highest nontoxic dose for subretinal PCNA-Rz was 10 microg/microl, whereas 100 microg/microl showed disturbance of pigmentation with corresponding histological changes of retinal photoreceptor loss and retinal pigment epithelium proliferation or irregularities. Subretinal injection of all three doses of 5-FU did not show any toxicity. Serial injections of a mixture of 1.5 microg/microl 5-FU with 10 microg/microl of PCNA-Rz was found to be safe in rat eyes. CONCLUSIONS: Subretinal injections of the combination of PCNA-Rz (10 microg/microl) and 5-FU (1.5 microg/microl) demonstrated to be safe in rat eyes during the course of this study, even with a multiple administration of four injections.

Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors.

Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.

Voltage-dependent structural interactions in the Shaker K(+) channel.

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K(+) channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D. M. Papazian. 1997. Biophys. J. 72:1489-1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K(+) channels.

Calnexin associates with Shaker K+ channel protein but is not involved in quality control of subunit folding or assembly.

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacted transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. We also investigated whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. Our results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels.

Shaker and ether-à-go-go K+ channel subunits fail to coassemble in Xenopus oocytes.

Members of different voltage-gated K+ channel subfamilies usually do not form heteromultimers. However, coassembly between Shaker and ether-à-go-go (eag) subunits, members of two distinct K+ channel subfamilies, was suggested by genetic and functional studies (Zhong and Wu. 1991. Science. 252: 1562-1564; Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We investigated whether Shaker and eag form heteromultimers in Xenopus laevis oocytes using electrophysiological and biochemical approaches. Coexpression of Shaker and eag subunits produced K+ currents that were virtually identical to the sum of separate Shaker and eag currents, with no change in the kinetics of Shaker inactivation. According to the results of dominant negative and reciprocal coimmunoprecipitation experiments, the Shaker and eag proteins do not interact. We conclude that Shaker and eag do not coassemble to form heteromultimers in Xenopus oocytes.

Subunit folding and assembly steps are interspersed during Shaker potassium channel biogenesis.

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 amino-terminal deletion, Delta97-196, 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 (alpha) and cytoplasmic beta subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. Delta97-196 subunits failed to establish any of these quaternary interactions. The Delta97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the beta 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.

Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits.

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.

Intersubunit interaction between amino- and carboxyl-terminal cysteine residues in tetrameric shaker K+ channels.

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.

Conserved cysteine residues in the shaker K+ channel are not linked by a disulfide bond.

Many voltage-activated K+ channels contain two conserved cysteine residues in putative transmembrane segments S2 and S6. It has been proposed that these cysteines form an intrasubunit disulfide bond [Guy, H.R., & Conti, F. (1990) Trends Neurosci. 13, 201-206]. This proposal was tested using site-directed mutagenesis followed by electrophysiological and biochemical analysis of the Shaker B K+ channel. Each Shaker B subunit contains seven cysteine residues, including the conserved residues C286 and C462 and a less conserved cysteine, C245. Each cysteine in the Shaker B protein can be mutated individually without eliminating functional activity, indicating that the protein does not contain a disulfide bond that is essential for protein folding or the assembly of active channels. To determine whether there is a nonessential disulfide bond, Shaker B protein was subjected to limited proteolysis. Fragments were analyzed by electrophoresis under reducing and nonreducing conditions followed by immunoblotting. The results indicate that the two conserved residues C286 and C462 do not form a disulfide bond with each other or with C245. In addition, the subunits are not linked by disulfide bonds. In HEK293T cells, Shaker B protein is first made as an incompletely glycosylated precursor that is converted to the fully glycosylated mature protein. Glycosylation occurs at two positions in the S1-S2 loop.