P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers.

BACKGROUND AND PURPOSE: The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH: We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS: Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONS: We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.

A protocol for low contamination risk of autologous serum drops in the management of ocular surface disorders.

AIM: To assess microbial contamination of 20% autologous serum (AS) eye drops used in a hospital inpatient setting. METHOD: 14 patients received autologous serum drops from 4 to 14 days with a cumulative total of 67 days. For each day the first and last drop (total 134 samples) was cultured on broth and blood agar. RESULTS: Four patients (9 samples) grew Staphylococcus epidermidis only. One patient (1 sample) showed Staphylococcus epidermidis and a scanty growth of viridans streptococci in the same sample, and on different days the same patient grew Staphylococcus aureus in one sample and Staphylococcus epidermidis in another sample. One patient (1 sample) grew micrococcus. There was no clinical or microbial evidence of infection in any of these six patients. CONCLUSION: This study shows that autologous serum drops can be safely used in an inpatient setting, under a strict protocol of preparation and storage, without significant risk of bacterial contamination and consequent infection.

Management options for the flat corneal graft.

The post-penetrating keratoplasty (PK) corneal profile may be unpredictable and visual rehabilitation can be difficult. A case series of 11 such patients with flat corneal topography is presented and their management described. Suture removal may not provide a sufficient re-modelling effect to restore convexity of the cornea to enable satisfactory contact lens fitting and reverse geometry (RG) lenses have an important role.

Xe991 reveals differences in K(+) channels regulating chloride secretion in murine airway and colonic epithelium.

The cognitive enhancer XE991 interacts with K(+) channels consisting of KCNQ2 and KCNQ3 heteromultimers to block the M-current. XE991 can also block KCNQ1 K(+) channels expressed in oocytes, but sensitivity is reduced when the channels are coexpressed with minK (KCNE1). The purpose of the study was to examine the interaction of XE991 with other types of K(+) channel, especially those in the basolateral membranes of murine epithelia. K(+) channel blockade was measured by the inhibition of chloride secretion resulting from depolarization. XE991 inhibited the chloride secretory current in colonic epithelia by an interaction with basolateral K(+) channels when forskolin was used as the stimulus. However, when 1-ethyl-2-benzimidazolinone (EBIO) was used to stimulate chloride secretion, XE991 was ineffective unless charybdotoxin was also present. Because EBIO also activates Ca(2+)-sensitive K(+) channels, whereas forskolin activates only cAMP-sensitive K(+) channels, it is concluded that the latter are the targets for XE991. XE991 had effects similar to those of 293B on epithelial chloride transport, for which the target is known to be KCNQ1/KCNE3 multimers. mRNA for both these components of the cAMP-sensitive K(+) channels were found in high abundance in the colon, whereas KCNE1 was barely detectable. Furthermore, both XE991 and 293B were active in colonic epithelia from KCNE1 knockout mice. By contrast, in nasal epithelium, the forskolin sensitive chloride secretory current was barely sensitive to XE991 but was sensitive to clofilium. Xenopus laevis oocytes in which both KCNQ1 and KCNE3 had been expressed were significantly more sensitive to XE991 than oocytes expressing only KCNQ1.

Long term clinical outcome of a randomised controlled trial of anterior chamber lenses after high volume intracapsular cataract surgery.

BACKGROUND: Age related cataract remains the major cause of blindness throughout the world. In many countries, the majority of cataract surgery continues to be done by the intracapsular cataract extraction (ICCE) method. The results of a large randomised controlled trial of multiflex open loop anterior chamber intraocular lenses (ACIOL) were reported from a busy eye hospital in Nepal. METHODS: There was a randomised controlled trial of 2000 people with bilateral cataract reducing vision to less than 6/36. Interventions were ICCE with an ACIOL compared with ICCE with aphakic spectacles (+11 dioptres). Participants were followed at discharge, 6 weeks, 1 year, and 1 1/2-5 years after surgery. Visual acuity and clinical outcome were measured. A poor outcome was defined as vision <6/60. RESULTS: Visual outcome was comparable in the two groups. More of the control group experienced functional blindness due to loss of aphakic spectacles at 1 year. The majority of cases of poor outcome occurred in the first year after surgery. There was no indication of any lens related problems after 1 year. CONCLUSION: Multiflex open loop anterior chamber lenses are safe for up to 1 year of follow up when used by experienced surgeons, and the available evidence of 2-5 years of follow up suggests that the complication rate is reasonably low. An ACIOL at the time of cataract surgery offers benefits over routine ICCE surgery with aphakic spectacle correction as it avoids the problem of replacing lost and broken spectacles.

Cerebral arteriovenous malformation presenting as visual deterioration in a child.

A rare case of visual loss as the presenting feature of a central arteriovenous malformation involving the vein of Galen is reported. A 5-year-old girl with a history of deteriorating vision for the past 6 months was examined. Ocular examination showed a left hemianopia, left optic atrophy, and dilated vessels of the right optic disc. MRI revealed a massive deep-seated central arteriovenous malformation involving the vein of Galen. The mechanism of visual loss is likely to be a combination of ischaemic optic atrophy associated with a steal phenomenon and direct compression of the right optic radiation.

Randomized controlled trial of anterior-chamber intraocular lenses in Nepal: long-term follow-up.

Most of the estimated 20 million people who are blind with cataracts live in rural areas of developing countries, where expert surgical resources are scarce. We have studied the use of multiflex open-loop anterior-chamber intraocular lenses (ACIOL) in high-volume low-cost surgery. Between 1992 and 1995, a total of 2000 people attending Lahan Eye Hospital, Nepal, with bilateral cataracts reducing vision to < or = 6/36 were randomly allocated to receive intracapsular extraction (ICCE) with aphakic spectacles, or ICCE with an ACIOL. We re-examined the cohort (1305/2000, 65%) between November 1996 and April 1997 and report the findings in this article. There were 13 new cases of poor visual outcome (best corrected vision < 6/60) arising after one year: 9 in the ACIOL group and 4 in the control group; odds ratio 2.1 (95% confidence interval, 0.59-9.55). The causes of poor outcome were as follows: ACIOL group--retinal detachment (4 cases), cystoid macular oedema (2), epiretinal membrane (1), age-related macular degeneration (1), and late endophthalmitis (1); control group--retinal detachment (2 cases), late endophthalmitis (1), and primary open-angle glaucoma with age-related macular degeneration (1). In rural areas of developing countries, well-manufactured multiflex open-loop ACIOLs can be implanted safely by experienced ophthalmologists after routine ICCE, avoiding the disadvantages of aphakic spectacle correction.

Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels.

1. G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gbetagamma and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1-GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1-GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4, 5-bisphosphate (PIP2). 2. The rate of inhibition of GIRK1-GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3. The dose-response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.

Identification of amino acids within the P2X2 receptor C-terminus that regulate desensitization.

1. The ATP-activated P2X2(a) and P2X2(b) receptor splice variants, which differ only in their C-terminal sequences, desensitize at different rates. We used mutational analysis to investigate the involvement of the C-terminal region in receptor desensitization. Rat wild-type and mutant P2X2 receptors were expressed in Xenopus oocytes and currents were measured using the two-electrode voltage-clamp technique. 2. Truncating P2X2 at the Lys369 splice site increased the rate of desensitization by >100-fold. Recovery from desensitization was slowed by approximately 5-fold. 3. Addition of Val370 onto the C-terminus of the truncated receptor slowed desensitization by approximately 70-fold. Point mutations that substituted either smaller or larger hydrophobic amino acids for Val370, within the P2X2(a) splice variant, had profound effects on the rate of desensitization. The rate decreased with increasing hydrophobicity but was not dependent upon the precise structure of the side group. 4. A mutant receptor, with only nine amino acids, Val-Asp-Pro-Lys-Gly-Leu-Ala-Gln-Leu, beyond the Lys369 splice site, desensitized at a similar rate to P2X2(a). Injection of the peptide of this sequence into oocytes expressing P2X2(a) increased the rate of desensitization, whereas the eight-residue peptide lacking the valine had no effect. 5. Neutralizing lysines in the vicinity of the splice site increased the rate of receptor desensitization. Substituting glutamine for Lys365 produced the greatest effect ( approximately 30-fold increase), whereas mutating lysines that were further upstream or downstream of this position had progressively less of an effect. 6. We conclude that the C-terminal splice site of the P2X2 receptor is located within a region that is critically involved in regulating the rate of receptor desensitization. The valine at position 370 interacts with an intracellular hydrophobic site to slow the rate of desensitization. Nearby lysines may facilitate this interaction.

Molecular determinants for sodium-dependent activation of G protein-gated K+ channels.

G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gbetagamma subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gbetagamma but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2 binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.

Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes.

1. The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein-gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild-type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2. Co-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1-Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine-induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4. Individually substituting the N- and C-terminal tails of Kir3.1 for those of Kir3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher ratio of ACh-induced to basal activity than the Kir3.1-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substituting the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core and C-terminal regions of Kir3.1 independently conferred time dependence on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.

Localization and functional expression of splice variants of the P2X2 receptor.

cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2 subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2 subunit were injected into Xenopus laevis oocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b) receptor were not significantly different from those at the P2X2(a) homomeric channel. However, the P2X2(b) receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a) receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b) receptor. When the P2X2(a) and P2X2(b) receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b) subunit. In situ hybridization of the three newly isolated isoforms of the P2X2 subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b) and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d) splice variant was present only in the nodose ganglion, at a low level.

Molecular determinants for assembly of G-protein-activated inwardly rectifying K+ channels.

Kir3.1 and Kir3.2 associate to form G-protein-activated, inwardly rectifying K+ channels. To identify regions involved in the coassembly of these subunits, truncated Kir3.1 polypeptides were coexpressed with epitope-tagged subunits in an in vitro translation system. N-terminal, C-terminal, and core region polypeptides were coimmunoprecipitated with both Kir3.2 and Kir3.1, suggesting that multiple elements distributed throughout the Kir3.1 polypeptide contribute to intersubunit binding interactions. The Kir3.2 C-terminal polypeptide coimmunoprecipitated with the Kir3.1 C-terminal polypeptide, but neither region recognized the N-terminal domain and core region of the Kir3.1 subunit. This suggests that within Kir3 channels the C-terminal domains of neighboring subunits interact. Coexpression of the truncated polypeptides with Kir3.1 and Kir3.2 in Xenopus oocytes reduced functional expression of the heteromeric channels. Constructs encoding the core region plus N-terminal and proximal C-terminal regions competed more effectively than the core region alone, which supports the contribution of all three regions to intersubunit binding interactions. Proximal and distal segments of the C-terminal domain were as effective at inhibiting functional expression as the entire C-terminal domain.

Atraumatic transfer of the donor corneal button.

PURPOSE: To devise a method for atraumatic transfer of the donor corneal button by using a corneal shovel. METHOD: The donor cornea is lifted with a microcorneal shovel, and the epithelial surface dried with a microsponge applied to the corneal shovel to create a seal. This allows the shovel to be rotated through 180 degrees over the host eye with no movement of the donor cornea. The seal is then broken by applying a drop(s) of fluid onto the epithelial surface of the donor cornea or by touching the edge of the donor cornea to the edge of the host cornea. The donor cornea then slides or is floated onto the host eye. RESULTS: This technique has been performed on 52 corneal transplants with no complications such as slippage or falling off of the donor corneal button during transfer. CONCLUSION: This method provides a useful atraumatic surgical procedure for stabilising and transferring the donor cornea during corneal transplantation.

Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides.

Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.