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Biomedical subjects

C A Gurnett

Publications and source records attributed to C A Gurnett.

17 recordsLinked to original sources
PubMed2001

Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel.

To investigate the molecular basis of the voltage sensor that triggers excitation-contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (alpha1S 1-670) and the III-IV domain (alpha1S 701-1873) were expressed in dysgenic alpha1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca(2+) transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Q(max) = 3 +/- 0.23 vs. 5.4 +/- 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V(1/2) = 0.2 +/- 3.5 vs. 22 +/- 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal alpha1A Ca(2+) channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the alpha1A I-II domain suggest that this hemi-Ca(2+) channel could be relevant to neuronal function.

Animals↗
PubMed2000

Localization of dominantly inherited isolated triphalangeal thumb to chromosomal region 7q36.

Triphalangeal thumb is an autosomal dominantly inherited form of abnormal preaxial skeletal development. In most families, however, the triphalangeal thumb phenotype coexists with a spectrum of limb deformities, including polydactyly and syndactyly. We describe two Iowa kindreds with triphalangeal thumb. In one family, with nine affected members, triphalangeal thumb was the only manifestation of limb deformity. We performed linkage analysis on both pedigrees, demonstrating a maximum LOD score of 6.23 with marker D7S559 on chromosome 7q36. This corresponds to a previous study of a candidate region of 450 kb in which data from several families with preaxial polydactyly were employed. Further analysis of the unique family with isolated triphalangeal thumb in the current study may demonstrate allelic variability of the gene involved in these disorders.

Chromosome Mapping↗
PubMed2000

Treatment of idiopathic clubfoot: an historical review.

Idiopathic clubfoot, one of the most common problems in pediatric orthopaedics, is characterized by a complex three-dimensional deformity of the foot. The treatment of clubfoot is controversial and continues to be one of the biggest challenges in pediatric orthopaedics. This controversy is due in part to the difficulty in measuring and evaluating the effectiveness of different treatment methods. We believe the heart of the debate is a lack of understanding of the functional anatomy of the deformity, the biological response of young connective tissue to injury and repair, and their combined effect on the long-term treatment outcomes. The aim of this review is not only to assess the different methods of clubfoot treatment used over the years in light of an evolving understanding of the pathoanatomy of the deformity, but to also clarify factors that allow a safe, logical approach to clubfoot management. Further research will be needed to fully understand the pathogenesis of clubfoot, as well as the long-term results and quality of life for the treated foot.

Clubfoot↗
PubMed1997

Dissection of functional domains of the voltage-dependent Ca2+ channel alpha2delta subunit.

Coexpression of the cloned voltage-dependent Ca2+ channel alpha2delta subunit with the pore-forming alpha1 subunit results in a significant increase in macroscopic current amplitude. To gain insight into the mechanism underlying this interaction, we have examined the regulatory effect of either the alpha2delta complex or the delta subunit on the Ca2+ channel alpha1 subunit. Transient transfection of tsA201 cells with the cardiac L-type alpha1C subunit alone resulted in the expression of inward voltage-activated currents as well as measurable [3H]-PN200-110 binding to membranes from transfected cells. Coexpression of the alpha2delta subunit significantly increased the macroscopic current amplitude, altered the voltage dependence and the kinetics of the current, and enhanced [3H]-PN200-110 binding. Except for the increase in amplitude, coexpression of the delta subunit reproduced entirely the effects of the full-length alpha2delta subunit on the biophysical properties of the alpha1C currents. However, no effect on specific [3H]-PN200-110 binding was observed on delta subunit coexpression. Likewise, profound effects on current kinetics of the neuronal alpha1A subunit were observed on coexpression of the alpha2delta complex in Xenopus oocytes. Furthermore, by using a chimeric strategy, we localized the region involved in this regulation to the transmembrane domain of the delta subunit. These data strongly suggest that the molecular determinants involved in alpha2delta regulation are conserved across L-type and non-L type Ca2+ channels. Taken together, our results indicate that the region of the alpha2delta subunit involved in the modulation of the gating properties of the high voltage-activated calcium channels is localized in the delta domain of the protein. In contrast, the level of membrane expression of functional channels relies on the presence of the alpha2 domain of the alpha2delta complex.

Calcium Channels↗
PubMed1997

Extracellular interaction of the voltage-dependent Ca2+ channel alpha2delta and alpha1 subunits.

The role of the extracellular domain of the voltage-dependent Ca2+ channel alpha2delta subunit in assembly with the alpha1C subunit was investigated. Transiently transfected tsA201 cells processed the alpha2delta subunit properly as disulfide linkages and cleavage sites between the alpha2 and delta subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of delta subunits, alpha2 subunits do not assemble with alpha1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in delta can be exchanged with those of an unrelated protein without any effect on the association between the alpha2delta and alpha1 proteins. Extracellular domains of the alpha2delta subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the alpha1C subunit. Investigation of the corresponding interaction site on the alpha1 subunit revealed that although tryptic peptides containing repeat III of native alpha1S subunit remain in association with the alpha2delta subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the alpha2delta subunit. Our results suggest that the alpha2delta subunit likely interacts with more than one extracellular loop of the alpha1 subunit.

Animals↗
PubMed1997

Direct binding of G-protein betagamma complex to voltage-dependent calcium channels.

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

Amino Acid Sequence↗
PubMed1997

Subunit stoichiometry of human muscle chloride channels.

Voltage-gated Cl- channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl- channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl- channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

Animals↗
PubMed1996

Expression and subunit interaction of voltage-dependent Ca2+ channels in PC12 cells.

Nerve growth factor (NGF)-induced differentiation in PC12 cells is accompanied by changes in the expression of voltage-dependent Ca2+ channels. Ca2+ channels are multimeric complexes composed of at least three subunits (alpha1, beta, and alpha2delta) and are involved in neuronal migration, gene expression, and neurotransmitter release. Although attempts have been undertaken to elucidate NGF regulation of Ca2+ channel expression, the changes in subunit composition of these channels during differentiation still remain uncertain. In the present study, patch-clamp recordings show that in addition to the previously documented L-type and N-type Ca2+ currents, undifferentiated PC12 cells also express an omega-agatoxin-IVA-sensitive (P/Q-type) component. In addition, the corresponding mRNA encoding the pore-forming alpha1 subunits for these channels (C, B, and A, respectively) was detected. Likewise, mRNA for three distinct auxiliary beta subunits (1, 2, 3) were also found, beta3 protein being dominantly expressed. Immunoprecipitation experiments show that the N-type Ca2+ channel is associated with either a beta2 or beta3 subunit and that NGF increases the channel expression without affecting its beta subunit association. These results (1) indicate that the diversity of Ca2+ currents in PC12 cells arise from the expression of three distinct alpha1 and three different beta subunit genes; (2) support a model for heterogenous beta subunit association of the N-type Ca2+ channel in a single cell type; and (3) suggest that the regulation of the N-type Ca2+ channel during NGF-mediated differentiation involves an increase in the number of functional channels with no apparent changes in subunit composition.

Animals↗
PubMed1996

Identification of three subunits of the high affinity omega-conotoxin MVIIC-sensitive Ca2+ channel.

N-, P- and Q-type voltage-dependent Ca2+ channels control neurotransmitter release in the nervous system and are blocked by omega-conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for omega-conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca2+ channel. Using optimized conditions for specific labeling of the high affinity omega-conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least alpha1A, alpha2delta, and any one of the four brain beta subunits. Such association of different beta subunits with alpha1A and alpha2delta components may produce Ca2+ channels with distinct functional properties, such as P- and Q-type.

Animals↗
PubMed1996

Beta subunit heterogeneity in N-type Ca2+ channels.

The beta subunit of the voltage-dependent Ca2+ channel is a cytoplasmic protein that interacts directly with an alpha1 subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the alpha1B subunit of the N-type Ca2+ channel associates with several different beta subunits. Polyclonal antibodies specific for three different beta subunits immunoprecipitated 125I-omega-conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca2+ channels with an alpha1B subunit-specific monoclonal antibody showed the association of beta1b, beta3, and beta4 subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca2+ channel confirmed the presence of the beta3 and beta4 subunits. Each of the beta subunits bound to the alpha1B subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the beta subunit composition of the N-type Ca2+ channels, which may be responsible for some of the diverse kinetic properties recorded from neurons.

Amino Acid Sequence↗
PubMed1996

Structural and functional diversity of voltage-activated calcium channels.

Data gathered from the expression of cDNAs that encode the subunits of voltage-dependent Ca2+ channels have demonstrated important structural and functional similarities among these channels. Despite these convergences, there are also significant differences in the nature and functional importance of subunit-subunit and protein-Ca2+ channel interactions. There is evidence demonstrating that the functional differences between Ca2+ channel subtypes is due to several factors, including the expression of distinct alpha 1 subunit proteins, the selective association of structural subunits and modulatory proteins, and differences in posttranslational processing and cell regulation. We summarize several avenues of research that should provide significant clues about the structural features involved in the biophysical and functional diversity of voltage-dependent Ca2+ channels.

Amino Acid Sequence↗
PubMed1996

Dual function of the voltage-dependent Ca2+ channel alpha 2 delta subunit in current stimulation and subunit interaction.

Voltage-dependent Ca2+ channels are modulated by complex interactions with the alpha 2 delta subunit. In vitro translation was used to demonstrate a single transmembrane topology of the alpha 2 delta subunit in which all but the transmembrane sequence and 5 carboxy-terminal amino acids are extracellular. The glycosylated extra-cellular domain is required for current stimulation, as shown by coexpression of truncated alpha 2 delta subunits with alpha 1A and beta 4 subunits in Xenopus oocytes and deglycosylation with peptide-N-glycosidase F. However, coexpression of the transmembrane domain-containing delta subunit reduced the stimulatory effects of full-length alpha 2 delta subunits and substitution of a different transmembrane domain resulted in a loss of current stimulation. These results support a model whereby the alpha 2 delta transmembrane domain mediates subunit interactions and the glycosylated extracellular domain enhances current amplitude.

Animals↗
PubMed1995

Absence of the skeletal muscle sarcolemma chloride channel ClC-1 in myotonic mice.

The voltage-dependent chloride channel ClC-1 stabilizes resting membrane potential in skeletal muscle. Mutations in the ClC-1 gene are responsible for both human autosomal recessive generalized myotonia and autosomal dominant myotonia congenita. To understand the tissue distribution and subcellular localization of ClC-1 and to evaluate its role in an animal model of myotonia, antibodies were raised against the carboxyl terminus of this protein. Expression of the 130-kDa ClC-1 protein is unique to skeletal muscle, consistent with its mRNA tissue distribution. Immunolocalization shows prominent ClC-1 antigen in the sarcolemma of both type I and II muscle fibers. Sarcolemma localization is confirmed by Western analysis of skeletal muscle subcellular fractions. The ADR myotonic mouse (phenotype ADR, genotype adr/adr), in which defective ClC-1 mRNA has been identified, is shown here to be absent in ClC-1 protein expression, whereas other skeletal muscle sarcolemma protein expression appears normal. Immunohistochemistry of skeletal muscle from ADR and other mouse models of human muscle disease demonstrate that the absence of ClC-1 chloride channel is a defect specific to ADR mice.

Amino Acid Sequence↗
PubMed1993

Norepinephrine release from guinea pig cardiac sympathetic nerves is insensitive to ryanodine under physiological conditions.

The activation of neurotransmitter release in nerve cells appears to be primarily dependent upon influx of extracellular Ca2+, most of which is thought to cross nerve terminal membranes through N-type Ca2+ channels. Events in skeletal and cardiac muscle, in contrast, are regulated to a greater extent by intracellular Ca2+ exchange between cytosol and intracellular organelles such as sarcoplasmic reticulum. It is not known to what extent corresponding intracellular organelles, i.e. endoplasmic reticulum (ER), contribute to cytosolic Ca2+ transients and norepinephrine (NE) release from cardiac sympathetic nerves. Heart rate and NE release were measured in isolated perfused guinea pig hearts during 1-min stimulations (5 V, 4 Hz, 2 ms) of the right stellate ganglia prior to (S1), during the administration of (S2), and after (S3) the removal of ryanodine (1 microM) from the perfusate. Ryanodine is a selective modulator of caffeine-sensitive Ca2+ stores in ER. Baseline heart rates decreased significantly in the presence of ryanodine, documenting its physiological effect on cardiac cells. However, there was no detectable effect of ryanodine on nerve-stimulated increase in heart rate or NE release. These results indicate that the ryanodine-sensitive intracellular Ca2+ stores do not play a major role in cardiac sympathetic neurotransmission.

Animals↗