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Drake C Mitchell

Publications and source records attributed to Drake C Mitchell.

13 recordsLinked to original sources
PubMed2005

Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density.

Rad, Gem/Kir, Rem, and Rem2 are members of the Ras-related RGK (Rad, Gem, and Kir) family of small GTP-binding proteins. Heterologous expression of RGK proteins interferes with de novo calcium channel assembly/trafficking and dramatically decreases the amplitude of currents arising from preexisting high-voltage-activated calcium channels. These effects probably result from the direct interaction of RGK proteins with calcium channel beta subunits. Among the RGK family, Rem2 is the only member abundantly expressed in neuronal tissues. Here, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-beta-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, [(125)I]Tyr22-omega-conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel beta subunits in the preassembled N-type channel, thereby forming a nonconducting species.

Animals↗
PubMed2005

Effect of packing density on rhodopsin stability and function in polyunsaturated membranes.

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.

Animals↗
PubMed2005

Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs.

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.

Animals↗
PubMed2005

Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein.

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.

Animals↗
PubMed2004

Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency.

The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.

Animals↗
PubMed2003

Enhancement of G protein-coupled signaling by DHA phospholipids.

The effect of phospholipid acyl chain and cholesterol composition on G protein-coupled signaling was studied in native rod outer segment (ROS) disk and reconstituted membranes by measuring several steps in the visual transduction pathway. The cholesterol content of disk membranes was varied from 4 to 38 mol% cholesterol with methyl-beta-cyclodextrin. The visual signal transduction system [rhodopsin, G protein (G(t)), and phosphodiesterase (PDE)] was reconstituted with membranes containing various levels of phospholipid acyl chain unsaturation, with and without cholesterol. ROS membranes from rats raised on n-3 fatty acid-deficient and -adequate diets were also studied. The ability of rhodopsin to form the active metarhodopsin II conformation and bind G(t) was diminished by a reduction in the level of DHA (22:6n-3) acyl chains or an increase in membrane cholesterol. DHA acyl chain containing phospholipids minimized the inhibitory effects of cholesterol on the rate of rhodopsin-G(t) coupling. The activity of PDE, which is a measure of the integrated signal response, was reduced in membranes lacking or deficient in DHA acyl chains. PDE activity in membranes containing docosapentaenoic acid (DPA, 22:5n-6) acyl chains, which replace DHA in n-3 fatty acid deficiency, was 50% lower than in DHA-containing membranes. Our results indicate that efficient and rapid propagation of G protein-coupled signaling is optimized by DHA phospholipid acyl chains.

Acylation↗
PubMed2003

DHA-rich phospholipids optimize G-Protein-coupled signaling.

OBJECTIVE: To assess the effects of n-3 polyunsaturated phospholipid acyl chains on the initial steps in G-protein-coupled signaling. STUDY DESIGN: Isolated components of the visual signal transduction system, rhodopsin, G protein (G(t)), and phosphodiesterase (PDE), were reconstituted in membranes containing various levels of n-3 polyunsaturated phospholipid acyl chains. In addition, rod outer segment disk membranes containing these components were purified from rats raised on n-3-deficient and n-3-adequate diets. The conformation change of rhodopsin, coupling of rhodopsin to G(t), and PDE activity were each measured separately. RESULTS: The ability of rhodopsin to form the active metarhodopsin II conformation and bind G(t) were both compromised in membranes with reduced levels of n-3 polyunsaturated acyl chains. The activity of PDE, directly related to the integrated cellular response, was reduced in all membranes lacking or deficient in n-3 polyunsaturated acyl chains. PDE activity in membranes containing 22:5n-6 PC was 50% lower than in membranes containing either 22:6n-3 PC or 22:5n-3 PC. CONCLUSIONS: The earliest events in G-protein-coupled signaling; receptor conformation change, receptor-G-protein binding, and PDE activity are reduced in membranes lacking n-3 polyunsaturated acyl chains. Efficient and rapid propagation of G-protein-coupled signaling requires polyunsaturated n-3 phospholipid acyl chains.

Fatty Acids, Omega-3↗
PubMed2002

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings.

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.

Animals↗
PubMed2002

Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation.

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.

Calorimetry, Differential Scanning↗
PubMed2002

Retinal sensitivity loss in third-generation n-3 PUFA-deficient rats.

A previous study conducted in guinea pigs suggested that ingestion of diets high in EPA and DHA may result in suboptimal retinal function. The aim of the present study was to evaluate retinal function in pigmented (Long-Evans) rats, raised to a third generation on diets that were either deficient in n-3 PUFA or adequate (with the addition of DHA). Electroretinographic assessment employed full-field white flash stimulation. Photoreceptor responses were evaluated in terms of peak amplitudes and implicit times (a-wave, b-wave), intensity-response functions (Naka-Rushton), and the parameters of a model of transduction (P3). Retinal phospholipid FA composition was measured by capillary GLC. DHA levels were reduced by 55% in n-3-deficient animals compared with the n-3-adequate group, whereas the levels of docosapentaenoic acid n-6 were 44 times higher in n-3-deficient animals. The level of arachidonic acid was marginally higher (12.8%) in n-6-adequate animals. The n-3-deficient animals exhibited significantly reduced retinal sensitivity (sigma and S values were both affected by 0.29 log units) and increased b-wave implicit times compared with those fed the n-3-adequate diet. These data suggest that n-3 PUFA are required for development of retinal sensitivity, more so than other indices of retinal function assessed by current methods, such as maximal response amplitude. However, the benefit for retinal function of adding preformed DHA to diets already replete in n-3 PUFA remains unclear.

Animals↗
PubMed2002

Visual acuity and retinal function in infant monkeys fed long-chain PUFA.

Previous randomized clinical trials suggest that supplementation of the human infant diet with up to 0.35% DHA may benefit visual development. The aim of the current study was to assess the impact of including arachidonic acid (AA) and a higher level of DHA in the postnatal monkey diet on visual development. Infant rhesus monkeys were fed either a control diet (2.0% alpha-linolenic acid as the sole n-3 FA) or a supplemented diet (1.0% DHA and 1.0% AA) from birth. Visual evoked potential acuity was measured at 3 mon of age. Rod and cone function were assessed in terms of parameters describing phototransduction. Electroretinogram (ERG) amplitudes and implicit times were recorded over a wide intensity range (-2.2 to 4.0 log scot td-sec) and assessed in terms of intensity response functions. Plasma DHA and AA were significantly increased (P < 0.001) in the diet-supplemented monkeys compared with the control monkeys. There was an approximately equal effect of diet for the rod phototransduction parameters, sensitivity, and capacitance but in the opposite directions. Diet-supplemented monkeys had significantly shorter b-wave implicit times at low retinal illuminances (<-0.6 log scot td-sec). There were no significant effects of diet for visual acuity or the other 23 ERG parameters measured. The results suggest that supplementation of the infant monkey diet with 1.0% DHA and 1.0% AA neither harms nor provides substantial benefit to the development of visual acuity or retinal function in the first four postnatal months.

Animals↗
PubMed2002

n-3 fatty acid deficiency alters recovery of the rod photoresponse in rhesus monkeys.

PURPOSE: Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid within rod outer segments. Varying the dietary content of DHA or its precursor, alpha-linolenic acid (ALA), can alter retinal DHA levels. The purpose of the present study was to assess rod phototransduction and recovery in rhesus monkeys raised on diets with different DHA and ALA content. METHODS: Adult rhesus monkeys had consumed from birth a diet low in ALA (0.3%) and known to induce an 80% reduction in retinal DHA. They were compared with groups receiving 8% ALA or 0.6% DHA, both of which support normal retinal DHA levels. Rod recovery was assessed with a double-flash protocol using a-wave saturating test flashes. The recovery of rod-isolated ERG a-wave amplitude was analyzed to determine Tc, the time to initiation of rod recovery, and T50, the time from initiation to 50% of full recovery. Phototransduction was assessed from the fit of a quantitative model to the leading edges of rod-isolated ERG a-waves. ERG a- and b-wave amplitudes and implicit times were also measured. RESULTS: Rod recovery (T50) was delayed by 30% and ERG implicit times by 5% in monkeys in the low ALA group compared with the other groups. There was no significant effect of diet on ERG amplitudes, the time to initiation of rod recovery, or the parameters describing phototransduction. CONCLUSIONS: The results indicate that mechanisms involved in deactivation and rod recovery are selectively altered in monkeys raised on a low-ALA diet whereas, at the flash intensities used, the mechanisms underlying phototransduction remain unaffected.

Animals↗