RESUMO
The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH(2)-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH(2)-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Células COS , Glicosilação , Proteínas de Membrana/genética , Dados de Sequência Molecular , Análise de SequênciaRESUMO
G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/metabolismo , Ressonância de Plasmônio de Superfície , Animais , Biotinilação , Bovinos , Ligantes , Rodopsina/metabolismoRESUMO
This paper describes the preliminary performance of a surgically implanted neuroprosthesis for standing and transfers after spinal cord injury (SCI) in an initial group of 12 volunteers with longstanding paralysis. The CWRU/VA standing neuroprosthesis consists of an 8-channel implanted receiver-stimulator, epimysial and surgically implanted intramuscular electrodes, and a programmable wearable external controller. After reconditioning exercise and rehabilitation with the system, most individuals with paraplegia or low tetraplegia were able to stand, transfer, and release one hand from a support device to manipulate objects in the environment or to perform swing-to ambulation in a walker. The effort and assistance required for transfers were reduced for users with mid-level tetraplegia, although the maneuvers were not independent. Neuroprosthesis users with tetraplegia and paraplegia alike benefited from the improvements in their general health derived from exercise, including reduced risk of decubiti and self-reported modulation of spasticity. Stimulated responses are stable and sufficiently strong for function, and implanted components are reliable with a 90% probability of epimysial electrode survival at 4 years post-implant. The techniques employed are repeatable and teachable, and suitable for multi-center clinical trial.
Assuntos
Terapia por Estimulação Elétrica , Paraplegia/reabilitação , Próteses e Implantes , Traumatismos da Medula Espinal/reabilitação , Eletrodos Implantados , Feminino , Humanos , Masculino , Postura , Desenho de PróteseAssuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/química , Transducina/metabolismo , Biofísica/métodos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Luz , Bicamadas Lipídicas , Fotólise , Espalhamento de Radiação , Espectrometria de Fluorescência/métodos , Espectrofotometria/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
OBJECTIVE: To determine the feasibility of providing the ability to stand and to facilitate the performance of standing transfers to individuals with cervical-level spinal cord injuries via functional neuromuscular stimulation (FNS). The applicability of implantable technology to this population was investigated, and the characteristics of the potential system users were explored. The effects of FNS on the effort and assistance required to stand and complete standing transfers were examined. SETTING: Institutional rehabilitation practice. DESIGN: Nonrandomized controlled trial. PATIENTS: Twenty-four individuals with low cervical spinal cord injuries were evaluated for inclusion in a program of lower extremity FNS, four of whom received the intervention. INTERVENTION: Chronically indwelling percutaneous intramuscular electrodes were used to exercise the hip, knee, and trunk extensors and develop activation patterns to produce standing function. These temporary systems were then replaced with silicone-enclosed helical wire electrodes suitable for eventual use with an eight-channel implantable receiver/stimulator. MAIN OUTCOME MEASURES: Full sensory and motor evaluations were performed and physical contraindications to stimulation were catalogued. For active subjects, American Spinal Injury Association Total Motor Scores with and without FNS were recorded, along with quadriceps strength and ability to complete exercise, standing, controlled sitting, and standing transfer maneuvers. Performances of implanted electrodes were determined by the stability of recruitment properties, impedances or surface potentials, and serial radiographs. RESULTS: Motor scores increased an average of nine points with stimulation over baseline volitional values. With FNS, all four volunteers were able to exercise, stand, and sit independently or with minimal assistance. Although they required varying degrees of assistance with the pivot phase of the transfer maneuver, all were able to raise and lower their body weight independently with stimulation and to use the system to facilitate standing transfers. One participant received the implantable receiver/stimulator, which remains operational at follow-up more than 3 years later. CONCLUSION: FNS can provide the ability to exercise, stand, and transfer to individuals with tetraplegia, even in the presence of medical complications and upper extremity impairment. FNS facilitates standing transfers by eliminating the heavy lifting usually required by a caregiver, thus decreasing the effort and assistance necessary to gain access to places impossible to approach with conventional sliding transfers.
Assuntos
Terapia por Estimulação Elétrica/métodos , Postura , Próteses e Implantes , Traumatismos da Medula Espinal/reabilitação , Adulto , Terapia por Estimulação Elétrica/instrumentação , Estudos de Viabilidade , Humanos , Masculino , Pessoa de Meia-Idade , Educação de Pacientes como Assunto , Desempenho Psicomotor , Resultado do TratamentoRESUMO
A 16-channel functional electrical stimulation (FES) system has been implanted in a person with T10 paraplegia for over a year. The system consists of two eight-channel radio frequency controlled receiver-stimulators delivering stimuli through a network of 14 epimysial and two intramuscular electrodes. Using this system and a walker for support, the subject was able to stand up for 8 min and walk regularly for 20 m. The standing duration was limited by arm fatigue since upper extremities supported an average of 25% of body weight. This was due to suboptimal hip extension and some undesired recruitment of rectus femoris and sartorius with stimulation of quadriceps electrodes. The left quadriceps exhibited rapid fatigue that limited walking distance and duration. The metabolic energy requirements were well within the aerobic limits of the sedentary paraplegic population. At one-year follow-up evaluation all electrodes are functional except one intramuscular electrode. The implant caused no adverse physiological effects and the individual reported health benefits such as increased energy and overall fitness as a result of the FES system use. With further improvements in muscle response through innovative surgical techniques, the 16-channel implanted FES system can be a viable addition to exercise and mobility function in persons with paraplegia.