ABSTRACT
Action potential generation is governed by the opening, inactivation, and recovery of voltage-gated sodium channels. A channel's voltage-sensing and pore-forming α subunit bears an intrinsic fast inactivation particle that mediates both onset of inactivation upon membrane depolarization and rapid recovery upon repolarization. We describe here a novel inactivation particle housed within an accessory channel subunit (A-type FHF protein) that mediates rapid-onset, long-term inactivation of several sodium channels. The channel-intrinsic and tethered FHF-derived particles, both situated at the cytoplasmic face of the plasma membrane, compete for induction of inactivation, causing channels to progressively accumulate into the long-term refractory state during multiple cycles of membrane depolarization. Intracellular injection of a short peptide corresponding to the FHF particle can reproduce channel long-term inactivation in a dose-dependent manner and can inhibit repetitive firing of cerebellar granule neurons. We discuss potential structural mechanisms of long-term inactivation and potential roles of A-type FHFs in the modulation of action potential generation and conduction.
Subject(s)
Fibroblast Growth Factors/pharmacology , Nerve Tissue Proteins/drug effects , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Animals , Antibodies, Blocking/pharmacology , Blotting, Western , Cell Line , Cerebellum/cytology , Cerebellum/physiology , Cytoplasm/drug effects , Cytoplasm/metabolism , Cytoplasmic Granules/physiology , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Electrophysiological Phenomena , Fibroblast Growth Factors/chemical synthesis , Fibroblast Growth Factors/immunology , Ion Channel Gating/drug effects , Mice , NAV1.6 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Reverse Transcriptase Polymerase Chain Reaction , Sodium Channels/genetics , TransfectionABSTRACT
Storage and release of endogenous growth factors by the extracellular matrix (ECM) are important biological events that control tissue homeostasis and regeneration. The interaction between basic fibroblast growth factor (bFGF) and heparan sulfate proteoglycans has been extensively studied and used as a prototype model of such a system, while the lower affinity of fibrillar type I collagen for bFGF has generally been considered biologically insignificant. However, our present investigation revealed that bFGF spontaneously interacts with type I collagen solution and sponges under in vitro and in vivo physiological conditions, and is protected from the proteolytic environment by the collagen. bFGF incorporated in a collagen sponge sheet was sustainedly released in the mouse subcutis according to the biodegradation of the sponge matrix, and exhibited local angiogenic activity in a dose-dependent manner. Intramuscular injection of collagen microsponges incorporating bFGF induced a significant increase in the blood flow in the murine ischemic hindlimb, which could never have been attained by bolus injection of bFGF. These results suggest the significance and therapeutic utility of type I collagen as a reservoir of bFGF.
Subject(s)
Collagen Type I/metabolism , Drug Carriers/metabolism , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacokinetics , Animals , Chemistry, Pharmaceutical/methods , Circular Dichroism/methods , Collagen/chemical synthesis , Collagen/metabolism , Collagen/pharmacology , Collagen Type I/chemical synthesis , Collagen Type I/drug effects , Dose-Response Relationship, Drug , Drug Carriers/chemistry , Drug Carriers/pharmacology , Female , Fibroblast Growth Factors/chemical synthesis , Hindlimb/anatomy & histology , Iodine Isotopes , Japan , Lower Extremity/blood supply , Mice , Mice, Inbred C57BL , Muscle, Skeletal/blood supply , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Neovascularization, Physiologic/drug effects , Neovascularization, Physiologic/physiology , Skin/chemistry , Skin/drug effects , Skin/metabolism , Trypsin/metabolismABSTRACT
Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.
Subject(s)
Fibroblast Growth Factor 1/chemistry , Fibroblast Growth Factors/chemical synthesis , Mitogens/chemical synthesis , Oligopeptides/chemical synthesis , Peptide Fragments/chemical synthesis , Peptides, Cyclic/chemical synthesis , 3T3 Cells , Animals , Circular Dichroism , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/pharmacology , Humans , Magnetic Resonance Spectroscopy , Mice , Mitogens/chemistry , Mitogens/pharmacology , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Conformation , Thymidine/metabolismABSTRACT
Peptides can be designed that mimic protein interaction motifs and thus, can be used to specifically and selectively block particular steps in signal transduction cascades where protein interactions have been previously identified. This protocol describes methods to synthesize peptides coupled to a membrane-permeable sequence (MPS), designed from the signal sequence of Kaposi fibroblast growth factor, which has been previously shown to translocate covalently attached cargo peptides across the cell membrane. To increase efficiency, yield, and versatility in the preparation of these membrane-permeable peptides, a modular synthesis strategy based on two unprotected peptide segments was designed. The modular synthesis strategy allows the MPS and functional peptides to be synthesized separately. In this manner, the functional domain of a peptide or protein, synthesized by traditional fluoroenylmethyloxy-carbonyl (Fmoc) chemistry or derived from recombinant expression, may be purchased commercially to expedite synthesis. Subsequently, the MPS domain may be attached to any functional domain using a one-step conjugation reaction. This protocol provides detailed methods for peptide synthesis, activation of the MPS, and the subsequent conjugation protocol.
Subject(s)
Peptides/chemistry , Peptides/chemical synthesis , Receptors, Cell Surface/chemistry , Signal Transduction , Animals , Binding Sites , Fibroblast Growth Factor 4 , Fibroblast Growth Factors/chemical synthesis , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/physiology , Humans , Protein Binding , Proto-Oncogene Proteins/chemical synthesis , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/physiology , Receptors, Cell Surface/physiology , Recombinant Proteins/chemistryABSTRACT
El factor de crecimiento neuronal (FCN), miembro de la familia de las neurotrofinas, es una proteína que desempeña un papel decisivo en la sobrevivencia y metabolismo de las neuronas colinérgicas del sistema nervioso central (SNC). Debido a que el sistema colinérgico del SNC ha sido involucrado en los procesos de aprendizaje y memoria, durante la última década se han puesto en práctica varios procedimientos experimentales con la finalidad de administrar el FCN en el SCN. Simultáneamente se han registrado importantes avances en el conocimiento de las características inherentes a la estructura así como a los mecanismos de acción y biosíntesis de esta molécula. La presente revisión intenta mostrar un panorama global acerca del estado actual de las investigaciones en torno a estos temas
