Stephen E. Ryan

Effect of Membrane Lipid Composition on the Conformational Equilibria of the Nicotinic Acetylcholine Receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590–24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.

Structural Effects of Neutral and Anionic Lipids on the Nicotinic Acetylcholine Receptor AN INFRARED DIFFERENCE SPECTROSCOPY STUDY

The effects of both neutral and anionic lipids on the structure of the nicotinic acetylcholine receptor (nAChR) have been probed using infrared difference spectroscopy. The difference between infrared spectra of the nAChR recorded using the attenuated total reflectance technique in the presence and absence of the neurotransmitter analog, carbamylcholine, exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon ligand binding and subsequent desensitization. This spectral map is essentially identical in difference spectra recorded from native, native alkaline-extracted, and affinity-purified nAChR reconstituted into either soybean asolectin or egg phosphatidylcholine membranes containing both neutral and anionic lipids. This result suggests both a similar structure of the nAChR and a similar resting to desensitized conformational change in each membrane environment. In contrast, difference spectra recorded from the nAChR reconstituted into egg phosphatidylcholine membranes lacking neutral and/or anionic lipids all exhibit an essentially identical pattern of band intensity variations, which is similar to the pattern of variations observed in difference spectra recorded in the continuous presence of the desensitizing local anesthetic, dibucaine. The difference spectra suggest that the main effect of both neutral and anionic lipids in a reconstituted egg phosphatidylcholine membrane is to help stabilize the nAChR in a resting conformation. In the absence of neutral and/or anionic lipids, the nAChR is converted into an alternate conformation that appears to be analogous to the local anesthetic-induced desensitized state. Significantly, the proportion of receptors found in the resting versus the putative desensitized state appears to be dependent upon the final lipid composition of the reconstituted membrane. A lipid-dependent modulation of the equilibrium between a channel-active resting and channel-inactive desensitized state may account for the modulations of nAChR activity that are observed in different lipid membranes.

Lipid composition alters drug action at the nicotinic acetylcholine receptor.

We tested the hypothesis that membrane lipid composition influences drug action at membrane proteins by studying local anesthetic action at the nicotinic acetylcholine receptor (nAChR). Infrared difference spectra show that concentrations of tetracaine consistent with binding to the ion channel (<50 μM) stabilize a resting-like state when the nAChR is reconstituted into phosphatidylcholine membranes containing the anionic lipid, phosphatidic acid, but have no effect on the nAChR reconstituted into membranes lacking phosphatidic acid, either in the presence or absence of cholesterol. Concentrations of tetracaine above 200 μM lead to neurotransmitter site binding in all membranes. In the presence of phosphatidic acid, cholesterol, or both, neurotransmitter site binding leads to the formation of quaternary amine-aromatic interactions between tetracaine and binding site tyrosine/tryptophan residues and the stabilization of a desensitized state. One interpretation suggested by lipid partitioning studies is that phosphatidic acid enhances tetracaine action at the channel pore by increasing the partitioning of tetracaine into the lipid bilayer, thereby enhancing access to the transmembrane pore. However, subtle membrane-dependent variations in the vibrations of tyrosine and tryptophan residues, and agonist analog binding studies indicate that the structures of the agonist-bound neurotransmitter sites of the nAChR in membranes lacking both phosphatidic acid and cholesterol differ from the structures of the agonist-desensitized neurotransmitter sites in the presence of both lipids. Lipid action at the nAChR thus involves more than a simple modulation of the equilibrium between resting and desensitized states.

Dissecting the Chemistry of Nicotinic Receptor-Ligand Interactions with Infrared Difference Spectroscopy

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm−1 indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm−1 are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm−1 indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm−1 to 1690 and 1620 cm−1, respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.

Randomized Trial Comparing the Primary Patency following Cutting Versus High-Pressure Balloon Angioplasty for Treatment of de Novo Venous Stenoses in Hemodialysis Arteriovenous Fistulae

PURPOSE A single-center randomized clinical trial was performed to compare postinterventional primary patency rates achieved by cutting balloon angioplasty and high-pressure balloon angioplasty in the treatment of de novo stenoses within autogenous arteriovenous (AV) fistulae for hemodialysis. MATERIALS AND METHODS Forty-eight patients undergoing their first angioplasty were prospectively randomized to undergo angioplasty with a cutting balloon or high-pressure balloon 4-8 mm in diameter because cutting balloons larger than 8 mm are not available. Nine patients were excluded after angiography, with seven requiring balloons larger than 8 mm. In the remaining 39 patients, there were 42 stenoses in the following regions: juxtaanastomotic (38%), perianstomotic (38%), midcephalic (9%), and cephalic arch (14%). Patients in the cutting balloon group were younger (mean age difference, 9 y; P = .04), but other demographic variables were comparable (range, P = .08-.89). The mean follow-up period was 8.5 mo (range, 24 d to 32 mo). Kaplan-Meier analysis was used to compare duration of patency. Mann-Whitney rank-sum t test and χ2/Fisher exact tests were used to compare continuous and categoric variables, respectively. RESULTS Technical success was achieved in all 39 patients. At 3, 6, and 12 months, the postinterventional primary patency rates for the cutting balloon group were 61.1% (95% confidence interval [CI], 35.75%-82.70%), 27.7% (95% CI, 9.69%-53.48%), and 11.1% (95% CI, 1.38%-34.71%), respectively, compared with 70.0% (95% CI, 45.72%-88.11%), 42.1% (95% CI, 20.25%-66.50%), and 26.3% (95% CI, 9.15%-51.20%), respectively, for the high-pressure balloon group (P < .3 at each interval). CONCLUSIONS Compared with high-pressure balloon angioplasty, cutting balloon angioplasty does not improve postinterventional primary patency of de novo stenotic lesions in autogenous arteriovenous fistulae.

Anesthetic-induced structural changes in the nicotinic acetylcholine receptor

The difference between infrared spectra of the nicotinic acetylcholine receptor (nAChR) recorded in the absence and presence of the agonist carbamylcholine (Carb) reveals a complex pattern of positive and negative bands that provides a spectral map of Carb-induced structural change. This spectral map is affected by the presence of either the local anesthetic, dibucaine, or the short chain alcohol, propanol. Both antagonists alter the intensities of difference bands in a manner consistent with the stabilization of a desensitized state. Spectral variations are also observed that are indicative of both the displacement of the anesthetics from the nAChR upon the addition of Carb and physical interactions that occur between the anesthetics and binding site residues.

Endoluminal dilatation for embedded hemodialysis catheters: A case-control study of factors associated with embedding and clinical outcomes

Background With the increasing frequency of tunneled hemodialysis catheter use there is a parallel increase in the need for removal and/or exchange. A small but significant minority of catheters become embedded or ‘stuck’ and cannot be removed by traditional means. Management of embedded catheters involves cutting the catheter, burying the retained fragment with a subsequent increased risk of infections and thrombosis. Endoluminal dilatation may provide a potential safe and effective technique for removing embedded catheters, however, to date, there is a paucity of data. Objectives 1) To determine factors associated with catheters becoming embedded and 2) to determine outcomes associated with endoluminal dilatation Methods All patients with endoluminal dilatation for embedded catheters at our institution since Jan. 2010 were included. Patients who had an embedded catheter were matched 1:3 with patients with uncomplicated catheter removal. Baseline patient and catheter characteristics were compared. Outcomes included procedural success and procedure-related infection. Logistic regression models were used to determine factors associated with embedded catheters. Results We matched 15 cases of embedded tunneled catheters with 45 controls. Among patients with embedded catheters, there were no complications with endoluminal dilatation. Factors independently associated with embedded catheters included catheter dwell time (> 2 years) and history of central venous stenosis. Conclusion Embedded catheters can be successfully managed by endoluminal dilatation with minimal complications and factors associated with embedding include dwell times > 2 years and/or with a history of central venous stenosis.

Externalization of Tunneled Hemodialysis Catheter in Patients with Tunnel or Exit-Site Infections and Limited Access Options

PURPOSE To evaluate the viability and effectiveness of temporary externalization of a tunneled hemodialysis (HD) catheter in catheter-dependent HD patients presenting with catheter-related tunnel or exit-site infection, documented central venous stenosis, and limited alternative venous access. MATERIALS AND METHODS All catheter-dependent HD patients with known central venous stenosis presenting with exit-site or tunnel infection and who subsequently underwent catheter externalization between February 2008 and May 2012 were reviewed. After catheter externalization, patients were concurrently treated with antibiotics for approximately 3 weeks before reinsertion of a new tunneled catheter. Treatment outcomes were collected, with treatment failures defined as reinfection with the same organism within 45 days of tunneled catheter reinsertion. RESULTS There were 42 catheter externalization procedures performed in 26 patients for 42 exit-site or tunnel infections. Technical success rate for catheter externalization was 100%, with no complications during the externalization procedure and preservation of all original access sites. Treatment failure occurred in 9.8% (4 of 41) of cases. Median infection-free survival after treatment and retunneling of a new dialysis catheter was 80 days. One major periprocedural complication of death occurred before reinsertion of a new tunneled catheter. Minor complications after the procedure occurred in four patients and included three cases of a small persistent wound at the temporary supraclavicular access site and one initially nonfunctioning externalized catheter. CONCLUSIONS Temporary dialysis catheter externalization appears both technically feasible and effective for the treatment of exit-site and tunnel infections, while allowing preservation of the venous access site in catheter-dependent HD patients with central venous stenosis and limited alternative venous access.