Margaret M. Briggs

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile585 being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.

The extent of amino-terminal heterogeneity in rabbit fast skeletal muscle troponin T

SummaryThe extent and nature of fast troponin T (TnT) heterogeneity has been assessed in rabbit skeletal muscle. Previous studies identified two major fast TnT species (TnT1f and TnT2f), in the fast white muscle erector spinae, differing in theirN-terminal cyanogen bromide (CNBr) fragments. Here a monoclonal antibody that recognizes a conserved region of TnT was used to characterize two additional TnT species (TnT3f and TnT4f) in the epaxial and limb musculature and a minor species (TnTcf) in craniofacial muscles. A combination of CNBr peptide mapping, immunoblotting and specific labelling of theN-terminus shows that these TnT species also differ in theirN-terminal region. This observation is consistent with cDNA studies that predicted theN-terminal region is hypervariable. One additional species, a variant of TnT2f present in the tongue, was identified by two-dimensional gel electrophoresis. The limited number of TnT variants indicates that the full potential for heterogeneity inferred from the cDNA studies is not realized. This conclusion is supported by immunoblot analysis with a monoclonal antibody that recognizes an epitope in the hypervariableN-terminal region which is present in all variants of TnT1f and TnT2f but absent from the lower molecular weight species TnT3f and TnT4f.

Water Permeability of Aquaporin-4 Channel Depends on Bilayer Composition, Thickness, and Elasticity

Aquaporin-4 (AQP4) is the primary water channel in the mammalian brain, particularly abundant in astrocytes, whose plasma membranes normally contain high concentrations of cholesterol. Here we test the hypothesis that the water permeabilities of two naturally occurring isoforms (AQP4-M1 and AQP4-M23) depend on bilayer mechanical/structural properties modulated by cholesterol and phospholipid composition. Osmotic stress measurements were performed with proteoliposomes containing AQP4 and three different lipid mixtures: 1), phosphatidylcholine (PC) and phosphatidylglycerol (PG); 2), PC, PG, with 40 mol % cholesterol; and 3), sphingomyelin (SM), PG, with 40 mol % cholesterol. The unit permeabilities of AQP4-M1 were 3.3 ± 0.4 × 10(-13) cm(3)/s (mean ± SE), 1.2 ± 0.1 × 10(-13) cm(3)/s, and 0.4 ± 0.1 × 10(-13) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. The unit permeabilities of AQP4-M23 were 2.1 ± 0.2 × 10(-13) cm(3)/s, 0.8 ± 0.1 × 10(-13) cm(3)/s, and 0.3 ± 0.1 × 10(-13) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. Thus, for each isoform the unit permeabilities strongly depended on bilayer composition and systematically decreased with increasing bilayer compressibility modulus and bilayer thickness. These observations suggest that altering lipid environment provides a means of regulating water channel permeability. Such permeability changes could have physiological consequences, because AQP4 water permeability would be reduced by its sequestration into SM:cholesterol-enriched raft microdomains. Conversely, under ischemic conditions astrocyte membrane cholesterol content decreases, which could increase AQP4 permeability.

Expression of a novel combination of fast and slow troponin T isoforms in rabbit extraocular muscles

SummaryThe properties of extraocular muscles (EOMs) are quite different from those of the trunk and limb. Here we show that there is a novel pattern of troponin T (TnT) expression in EOMs which most likely contributes to the fine control of ocular movement and may reflect their innervation by cranial motoneurons. Three regions of the muscle were analysed to distinguish the TnT isoforms present in the fast singly-innervated fibres from those in the multiply-innervated fibres. More than 95% of the TnT in the singly-innervated fibres is TnT3f, which exhibits the most graded response to changes in calcium concentration during activation (Schachatet al., J. molec. Biol.198, 551–4). In multiply-innervated fibres, which exhibit tonic contractures, the slow troponin T TnT2s is expressed. While neither TnT3f nor TnT2s is unique to EOM, this pattern is unusual in two respects: first, both TnT3f and TnT2s are minor components of the trunk and limb musculature, and second, most muscles express several fast and both slow TnT species. Although EOM occupies a highly specialized physiological niche, its unusual physiology is not reflected in the presence of new TnT isoforms but in the expression of a different ratio of the known species of TnT.

The water permeability of lens aquaporin-0 depends on its lipid bilayer environment.

Aquaporin-0 (AQP0), the primary water channel in lens fiber cells, is critical to lens development, organization, and function. In the avascular lens there is thought to be an internal microcirculation associated with fluid movement. Although AQP0 is known to be important in fluid fluxes across membranes, the water permeability of this channel has only been measured in Xenopus oocytes and in outer lens cortical membranes, but not in inner nuclear membranes, which have an increased cholesterol/phospholipid ratio. Here we measure the unit water permeability of AQP0 in different proteoliposomes with cholesterol/phospholipid ratios and external pHs similar to those found in the cortex and nucleus of the lens. Osmotic stress measurements were performed with proteoliposomes containing AQP0 and three different lipids mixtures: (1) phosphatidylcholine (PC) and phosphatidylglycerol (PG), (2) PC, PG, with 40 mol% cholesterol, and (3) sphingomyelin (SM), PG, with 40 mol% cholesterol. At pH 7.5 the unit permeabilities of AQP0 were 3.5 ± 0.5 × 10(-14) cm(3)/s (mean ± SEM), 1.1 ± 0.1 × 10(-14) cm(3)/s, and 0.50 ± 0.04 × 10(-14) cm(3)/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. For lipid mixtures at pH 6.5, corresponding to conditions found in the lens nucleus, the AQP0 permeabilities were 1.5 ± 0.4 × 10(-14) cm(3)/s and 0.76 ± 0.03 × 10(-14) cm(3)/s in PC:PG:cholesterol and SM:PG:cholesterol, respectively. Thus, although AQP0 unit permeability can be modified by changes in pH, it is also sensitive to changes in bilayer lipid composition, and decreases with increasing cholesterol and SM content. These data imply that AQP0 water permeability is regulated by bilayer lipid composition, so that AQP0 permeability would be significantly less in the lens nucleus than in the lens cortex.

Sorting of Lens Aquaporins and Connexins into Raft and Nonraft Bilayers: Role of Protein Homo-Oligomerization

Two classes of channel-forming proteins in the eye lens, the water channel aquaporin-0 (AQP-0) and the connexins Cx46 and Cx50, are preferentially located in different regions of lens plasma membranes (1,2). Because these membranes contain high concentrations of cholesterol and sphingomyelin, as well as phospholipids such as phosphatidylcholine with unsaturated hydrocarbon chains, microdomains (rafts) form in these membranes. Here we test the hypothesis that sorting into lipid microdomains can play a role in the disposition of AQP-0 and the connexins in the plane of the membrane. For both crude membrane fractions and proteoliposomes composed of lens proteins in phosphatidylcholine/sphingomyelin/cholesterol lipid bilayers, detergent extraction experiments showed that the connexins were located primarily in detergent soluble membrane (DSM) fractions, whereas AQP-0 was found in both detergent resistant membrane and DSM fractions. Analysis of purified AQP-0 reconstituted in raft-containing bilayers showed that the microdomain location of AQP-0 depended on protein/lipid ratio. AQP-0 was located almost exclusively in DSMs at a 1:1200 AQP-0/lipid ratio, whereas approximately 50% of the protein was sequestered into detergent resistant membranes at a 1:100 ratio, where freeze-fracture experiments show that AQP-0 oligomerizes (3). Consistent with these detergent extraction results, confocal microscopy images showed that AQP-0 was sequestered into raft microdomains in the 1:100 protein/lipid membranes. Taken together these results indicate that AQP-0 and connexins can be segregated in the membrane by protein-lipid interactions as modified by AQP-0 homo-oligomerization.

Patterns of troponin T expression in mammalian fast, slow and promiscuous muscle fibres.

SummaryThe distribution of troponin T(TnT) species in typed single muscle fibres was analysed using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and a monoclonal antibody specific for fast TnT. Fibres taken from erector spinae (Es), plantaris (Plt), diaphragm (Dia) and soleus (Sol) muscles of adult rabbits were pretyped as fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), slow-twitch-oxidative (SO) or promiscuous (P) using a combination of histochemical staining and PAGE. Although none of the four size classes of TnT was either muscle or fibre type specific, their pattern of expression differed in each muscle and between the fibre types. FG fibres expressed TnT1f or TnT2f as predominant species, depending on the muscle; TnT3f and TnT4f were minor components. In contrast, all size classes of TnT were expressed in varying proportions in FOG fibres from Es and Plt, while those from Dia resembled FG fibres, expressing TnT1f as their major species. P fibres from Es, Plt, and Sol exhibited a distinctive pattern of fast TnT expression, TnT3f being the predominant species. Dia differed from the other muscles as TnT1f was the dominant fast TnT species in its P fibres as it is in the Dia fast fibres. Quantitative analysis of one- and two-dimensional gels revealed that the P fibres could be divided into two classes, those that exhibited discoordinate expression of fast and slow TnTs, myosin light chains and myosin heavy chains and those in which their expression was coordinate. In addition low levels of TnT4f were detected in SO fibres and of slow TnT in fast fibres.

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and α-actinin isoforms in developing rabbit skeletal muscle

In adult fast skeletal muscle, specific combinations of thin filament and Z-line protein isoforms are coexpressed. To determine whether the expression of these sets of proteins, designated the TnT1f, TnT2f, and TnT3f programs, is coordinated during development, we characterized the transitions in troponin T (TnT), tropomyosin (Tm), and alpha-actinin isoforms that occur in developing fetal and neonatal rabbit skeletal muscle. Two coordinated developmental transitions were identified, and a novel pattern of thin filament expression was found in fetal muscle. In fetal muscle, new TnT species--whose protein and immunochemical properties suggest that they are the products of a new TnT gene--are expressed in combination with beta 2 Tm and alpha-actinin1f/s. This pattern, which is found in both back and hindlimb muscles, is specific to fetal and early neonatal muscle. Just prior to birth, there is a transition from the fetal program to the isoforms that define the TnT3f program, TnT3f, and alpha beta Tm. Like the fetal program, expression of the TnT3f program appears to be a general feature of muscle development, because it occurs in a variety of fast muscles as well as in the slow muscle soleus. The transition to adult patterns of thin filament expression begins at the end of the first postnatal week. Based on studies of erector spinae, the isoforms comprising the TnT2f program, TnT2f, alpha 2 Tm, and alpha-actinin2f, appear and increase coordinately at this time. The transitions, first to the TnT3f program, and then to adult patterns of expression indicate that synthesis of the isoforms comprising each program is coordinated during muscle specialization and throughout muscle development. In addition, these observations point to a dual role for the TnT3f program, which is the major thin filament program in some adult muscles, but appears to bridge the transition from developmentally to physiologically regulated patterns of thin filament expression during the late fetal and early neonatal development.

Identification of a fetal exon in the human fast Troponin T gene

A developmentally regulated exon has been identified in the 5′‐alternatively spliced region of the human fast Troponin T (TnT) gene. Expressed in fetal (but not adult) muscle, this exon is homologous with the fetal exons recently described in the rabbit and rat fast TnT genes. They all exhibit a split codon organization and encode a highly acidic peptide. To determine if the splicing pathways, including the human fetal exon, are also conserved, we defined the major TnT splicing patterns in fetal muscle. They generate fetal TnT 1, fetal TnT 3, and TnT1f, and TnT3f, species previously described in rabbit and rat skeletal muscles.

Regional variation in IIM myosin heavy chain expression in the temporalis muscle of female and male baboons (Papio anubis)

OBJECTIVE The purpose of this study was to determine whether high amounts of fast/type II myosin heavy chain (MyHC) in the superficial as compared to the deep temporalis muscle of adult female and male baboons (Papio anubis) correlates with published data on muscle function during chewing. Electromyographic (EMG) data show a regional specialization in activation from low to high amplitude activity during hard/tough object chewing cycles in the baboon superficial temporalis.(48,49) A positive correlation between fast/type II MyHC amount and EMG activity will support the high occlusal force hypothesis. DESIGN Deep anterior temporalis (DAT), superficial anterior temporalis (SAT), and superficial posterior temporalis (SPT) muscle samples were analyzed using SDS-PAGE gel electrophoresis to test the prediction that SAT and SPT will show high amounts of fast/type II MyHC compared to DAT. Serial muscle sections were incubated against NOQ7.5.4D and MY32 antibodies to determine the breadth of slow/type I versus fast/type II expression within each section. RESULTS Type I and type IIM MyHCs comprise nearly 100% of the MyHCs in the temporalis muscle. IIM MyHC was the overwhelmingly predominant fast MyHC, though there was a small amount of type IIA MyHC (≤5%) in DAT in two individuals. SAT and SPT exhibited a fast/type II phenotype and contained large amounts of IIM MyHC whereas DAT exhibited a type I/type II (hybrid) phenotype and contained a significantly greater proportion of MyHC-I. MyHC-I expression in DAT was sexually dimorphic as it was more abundant in females. CONCLUSIONS The link between the distribution of IIM MyHC and high relative EMG amplitudes in SAT and SPT during hard/tough object chewing cycles is evidence of regional specialization in fibre type to generate high occlusal forces during chewing. The high proportion of MyHC-I in DAT of females may be related to a high frequency of individual fibre recruitment in comparison to males.