Biomedical subjects

B H White

Publications and source records attributed to B H White.

At least 19 recordsLinked to original sources

PubMed2001

A conditional tissue-specific transgene expression system using inducible GAL4.

In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissue-specific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or "larval bathing." Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre- and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

Animals↗

PubMed2001

Targeted attenuation of electrical activity in Drosophila using a genetically modified K(+) channel.

We describe here a general technique for the graded inhibition of cellular excitability in vivo. Inhibition is accomplished by expressing a genetically modified Shaker K(+) channel (termed the EKO channel) in targeted cells. Unlike native K(+) channels, the EKO channel strongly shunts depolarizing current: activating at potentials near E(K) and not inactivating. Selective targeting of the channel to neurons, muscles, and photoreceptors in Drosophila using the Gal4-UAS system results in physiological and behavioral effects consistent with attenuated excitability in the targeted cells, often with loss of neuronal function at higher transgene dosages. By permitting the incremental reduction of electrical activity, the EKO technique can be used to address a wide range of questions regarding neuronal function.

Adaptation, Physiological↗

PubMed2000

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Animals↗

PubMed1999

Inhibition of hormonally induced inositol trisphosphate production in Transfected GH4</ sup>C1 cells: A novel role for the D5 subtype of the dopamine receptor.

We have previously found that the D5 dopamine receptor couples to a G-protein other than Gsalpha, and could be involved in signaling pathways other than regulation of adenylyl cyclase. To describe interactions of the D5 receptor with cellular effectors, we used GH4C1 cells transfected with cDNA for the human D5 receptor. Thyrotropin-releasing hormone (TRH, 100 nM) stimulated accumulation of inositol phosphates (IPs) fivefold in D5GH4C1 cells. Dopamine (DA, 10 microM) inhibited TRH-stimulated IP values by 29%; at higher concentrations (100 microM), maximal inhibition of 61% was observed. The D5 agonist SKF R-38393 (10 microM) mimicked this effect (28% inhibition). SCH 23390, a D5 antagonist, blocked the inhibition caused by both DA and SKF R-38393. Spiperone, a D2 receptor antagonist, did not block the inhibition. The D2 agonist (+/-)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin (PPHT) did not inhibit TRH-stimulated IP production, nor did it augment the effect of D5 agonists. The DA-mediated suppression of IP levels was not sensitive to pertussis toxin; cholera toxin blocked both TRH stimulation and DA suppression of IP accumulation in response to 100 nM TRH. Neither dibutyryl cAMP nor forskolin lowered IP formation in response to TRH. Phorbol ester decreased TRH-stimulated IP accumulation in D5GH4C1 cells; however, an inhibitor of protein kinase C (PKC) did not block the effect of DA.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1998

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz.

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.

Animals↗

PubMed1998

Alteration of association of agonist-activated renal D1(A) dopamine receptors with G proteins in proximal tubules of the spontaneously hypertensive rat.

BACKGROUND: Defective D1A dopamine receptor-G protein coupling has been identified in renal proximal tubules of the spontaneously hypertensive rat (SHR). OBJECTIVE: To determine whether association of D1A dopamine receptors with the alpha subunits of G proteins in kidney of SHR is normal. METHODS: We analyzed the association of agonist-activated [1251]-labeled D1A dopamine receptors in kidneys of SHR and the normotensive Wistar-Kyoto (WKY) rat through immunoprecipitation, using highly specific antipeptide antibodies directed against alpha subunits of G proteins. RESULTS: We have shown for the first time that the D1A receptors of renal proximal tubules are associated with the adenylyl cyclase inhibitory G proteins G(i)alpha. The association of WKY rat proximal tubule D1A receptors with Gi1alpha and Gi2alpha in the presence of agonist is significantly (P<0.01) greater (2.4-fold and 3.1-fold greater, respectively) than it is without agonist D1A receptors of WKY rat also exhibit (twofold greater) association with G(s)alpha, consistently with the ability of these receptors to mediate stimulation of adenylyl cyclase. The WKY rat D1A receptors do not associate either with G(o)alpha or with G(q)alpha. The D1A receptors of SHR proximal tubule membranes appear to be resistant to activation by agonist and do not associate with G(s)alpha, G(o)alpha and any of the subunits of G(i)alpha. However, the SHR D1A sites exhibit a modestly (1.7-fold) greater association with G(q)alpha, which was not statistically significant. The differences among associations of the D1A receptors of WKY rat and SHR with these Galpha proteins may be important in understanding renal dopaminergic functions in normal and pathophysiologic states.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1998

Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling.

AIM: Defective dopamine D1A dopamine receptor/G-protein coupling has been demonstrated in renal proximal tubules of the spontaneously hypertensive rat (SHR). In the present study, we aimed to analyze the underlying mechanisms through which such defects are introduced into the D1A receptor protein of SHR. MATERIALS AND METHODS: The oxidative state of SHR proximal tubules was analyzed by measuring lipid peroxidation. D1A receptor/G-protein coupling was measured following the induction of oxidative stress in normotensive Wistar-Kyoto (WKY) rats. RESULTS: For the first time, an increased state of oxidative stress was demonstrated in SHR proximal tubules compared with those of normotensive controls, WKY and Sprague-Dawley rats. Lipid peroxidation levels in SHR were significantly higher by 66 and 79%, relative to WKY or Sprague-Dawley rats, respectively. Hydrogen peroxide treatment of proximal tubules from SHR, WKY and Sprague-Dawley rats induced an additional increase in lipid peroxidation in a dose-dependent manner, although the percentage induction was lower in SHR than in WKY and Sprague-Dawley rats. This induction of lipid peroxidation in WKY rats resulted in a loss of D1A/G-protein coupling, with no decrease in receptor protein. Treatment of WKY rat proximal tubules with an antioxidant, ascorbic acid, or a reducing agent, dithiothreitol, induced D1A receptor/G-protein coupling. CONCLUSIONS: These data indicate that D1A receptor/G-protein coupling is modulated by changes in redox states. Therefore, the D1A receptor/G-protein coupling in SHR may have been damaged by reactive oxygen species released as a result of the elevated oxidative stress seen in the proximal tubules.

Animals↗

PubMed1998

Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of aplysia.

Protein kinase C (PKC) acutely increases calcium currents in Aplysia bag cell neurons by recruiting calcium channels different from those constitutively active in the plasma membrane. To study the mechanism of PKC regulation we previously identified two calcium channel alpha1-subunits expressed in bag cell neurons. One of these, BC-alpha1A, is localized to vesicles concentrated primarily in somata and growth cones. We used antibodies to BC-alpha1A to analyze its expression in the bag cell neurons of juvenile Aplysia at a developmental stage at which PKC-sensitive calcium currents have previously been shown to be low. We find that vesicular BC-alpha1A staining is generally reduced in juvenile bag cell neurons but that its expression level can vary among juvenile animals. In 17 bag cell clusters examined, the percentage of neurons that displayed punctate alphaBC-alpha1A staining ranged from 0 to 85%. Sampling of calcium currents from cells of the same clusters by whole cell patch-clamp techniques revealed that the PKC-sensitive calcium current density is significantly correlated with the degree of vesicular staining. In contrast, no correlation of basal calcium current levels with aBC-alpha1A staining was found. These results strongly suggest that BC-alpha1A, a member of the ABE-subfamily of calcium channels, carries the PKC-sensitive calcium current in bag cell neurons. They are consistent with a model in which PKC recruits channels from the vesicular pool to the plasma membrane.

Animals↗

PubMed1997

Identification of a vesicular pool of calcium channels in the bag cell neurons of Aplysia californica.

To study the molecular mechanism of calcium current modulation in the bag cell neurons of Aplysia californica, we have identified calcium channel subtypes expressed in these cells and analyzed their distribution using channel-specific antibodies. Using PCR to amplify reverse-transcribed RNA from bag cell clusters, we identified two classes of calcium channel alpha1 subunit. One, BCCa-I, belongs to the ABE subfamily of calcium channels, whereas the other, BCCa-II, belongs to the SCD subfamily. Antibodies generated against the bag cell calcium channels recognize membrane proteins of approximately 210 and 280 kDa on immunoblots. Both channels are expressed in the bag cell clusters as well as in other parts of the Aplysia nervous system. BCCa-II also localizes to glia and muscle. The subcellular distribution of the two channel types is strikingly different. Antibody staining of bag cell neurons in primary culture shows that BCCa-II is present on the plasma membrane, whereas BCCa-I has a punctate, intracellular distribution consistent with a vesicular localization. The BCCa-I-containing vesicles are found in bag cell neuron somata and growth cones and occasionally in neuritic hotspots. Their distribution is similar but not identical to that of LysoTracker Red, a marker for acidic organelles, but unlike that of dense-core vesicles containing egg-laying hormone. The vesicular channels may represent the protein kinase C-sensitive calcium channels of bag cell neurons that are believed to enhance hormonal release during electrical activity.

Amino Acid Sequence↗

PubMed1997

Molecular and structural differences between rat brain D-1 and renal DA-1 dopamine receptors.

Renal DA-1 dopamine receptors in proximal tubules (PTs) of the Wistar-Kyoto (WKY) rat display pharmacological binding properties which are different from central nervous system (CNS) striatal D-1 dopamine receptors. In general, the renal DA-1 receptors display affinity binding values of dopaminergic drugs which are 6-36-fold less than those seen for brain D-1 receptors. The renal and brain DA receptors also displayed differential sensitivity toward the alkylating agent, N-ethylmaleimide (NEM). Inactivation of 50% of DA-1 renal receptors was achieved at lower concentrations of NEM (5.2 microM), relative to brain D-1 receptors (140 microM). Western blot analyses of rat pituitary GH4C1 cells, transfected with human CNS D-1 receptor cDNA, with human anti-D-1 dopamine receptor antiserum, detected a single polypeptide with M(r) of 66 kDa. In PTs, a specific polypeptide of higher molecular weight (M(r) = 72 kDa) was seen. Surprisingly, in rat striatal membranes, the D-1 antiserum failed to detect any proteins within this molecular weight range. Photoaffinity labeling studies with a DA-1 selective photoligand, identified the identical protein by autoradiography and Western blots in kidney, but not in striate. Together, these data indicate that renal DA-1 dopamine receptors have distinct molecular properties relative to brain D-1 dopamine receptors.

Animals↗

PubMed1997

Dysfunctional D1A receptor-G-protein coupling in proximal tubules of spontaneously hypertensive rats is not due to abnormal G-proteins.

BACKGROUND: Dysfunctional dopamine neurotransmission and defective D1A receptor-G protein coupling exist in renal proximal tubules (RPT) of the spontaneously hypertensive rat (SHR). OBJECTIVE: To determine whether the G proteins in SHR are abnormal, preventing formation of agonist high affinity sites in SHR. METHODS: We examined the expression levels of the alpha-subunits of G proteins, as well as D1A receptor receptor coupling to exogenously added normal G proteins, in RPT of SHR and the normotensive Wister-Kyoto (WKY) rat. RESULTS: In the presence of 110 mmol/l NaCl, the D1A dopamine receptor-selective agonist SKF R-38393 binds both to high- and to low-affinity sites on solubilized and reconstituted D1A receptors extracted from renal proximal tubules of normotensive Wistar-Kyoto (WKY) rats. In the spontaneously hypertensive rat (SHR), SKF R-38393 bound to a single site on the reconstituted receptor with affinity values corresponding to the low-affinity state of the receptor. Western blot analyses indicated that the alpha-subunit of the guanine nucleotide binding protein (G-protein), Gs, was expressed at similar levels, whereas G(o)alpha was not expressed in proximal tubule membranes from WKY rats and SHR. Pretreatment of proximal tubule membranes with the alkylating agent N-ethylmaleimide in the presence of SKF R-38393 inactivated alpha-subunits of endogenous G-proteins, but not D1A receptors, resulting in loss of high-affinity binding sites in WKY rats. These N-ethylmaleimide-treated D1A receptors from WKY rats, when reconstituted with exogenous sources of G-proteins, were able to couple to these exogenous G-proteins, with complete restoration of high-affinity sites. Moreover, the affinity values and the proportion of these hybrid sites were similar to those of untreated receptors, and these affinity sites were regulated by guanine nucleotide analogs. Reconstitution of D1A receptors from SHR with the same exogenous G-proteins failed to similarly induce formation of the high-affinity binding sites in the hybrid reconstituted systems, and SKF R-38393 continued to bind in a single low-affinity state of the receptor. CONCLUSION: These results demonstrate that the absence of G-protein coupling in SHR is due to intrinsic defects within the receptor protein, rather than to any abnormalities of the endogenous G-proteins themselves.

Alkylating Agents↗

PubMed1996

CGP 39653 binding in the chick CNS after NMDA receptor antagonist treatment.

To analyze the affect of blocking neuronal activity on NMDA receptor levels during development, we have injected chick embryos with the competitive NMDA receptor antagonist NPC 12626 from E17 to E19. Brains from drug-treated (n = 7) and control (n = 6) embryos (E20) were processed for receptor autoradiography using the NMDA competitive antagonist [3H]CGP 39653. NPC 12626 treatment caused a significant 24 to 46% increase in [3H]CGP 39653 binding in both the forebrain and cerebellar cortex. The results support the hypothesis that NMDA receptor levels are regulated by activity-dependent mechanisms.

2-Amino-5-phosphonovalerate↗

PubMed1996

Increased expression of inhibitory guanine nucleotide binding proteins in membranes from renal proximal tubules, but not brain striata, of the normotensive and the spontaneously hypertensive rat.

Animals↗

PubMed1995

Coupling of human D-1 dopamine receptors to different guanine nucleotide binding proteins. Evidence that D-1 dopamine receptors can couple to both Gs and G(o).

Coupling between D-1 dopamine receptors and G proteins in cell lines expressing human D-1 receptors and different G proteins was examined. Pertussis toxin (PTX) treatment of rat pituitary GH4C1 cells significantly reduced, but did not abolish, agonist high affinity binding sites of the D-1 dopamine receptor; in SK-N-MC neuroblastoma cells, PTX failed to have any effect on D-1 high affinity sites. Cholera toxin (CTX) treatment of GH4C1 cells reduced but did not abolish the high affinity sites of D-1 receptors, while in SK-N-MC cells, treatment with CTX abolished all the high affinity sites. Western blot analyses with specific antisera indicated that Gs alpha, Gi1 alpha, Gi3 alpha, and Gq alpha were expressed in both cell lines, while Gi2 alpha and G(o) alpha were expressed in GH4C1 but not SK-N-MC cells. Antisera NEI-805 (anti-Gs alpha) and 9072 (anti-G(o) alpha) immunoprecipitated 24 +/- 4.3 and 34.4 +/- 6.9%, respectively, of G protein-associated D-1 dopamine receptors. Antisera 3646 (anti-Gi1 alpha), 1521 (anti-Gi2 alpha), 1518 (anti-Gi3 alpha), and 0941 (anti-Gq alpha) failed to coimmunoprecipitate appreciable levels of soluble receptors. These data indicate that D-1 dopamine receptors are coupled to both Gs alpha and G(o) alpha but not to Gq alpha.

Animals↗

PubMed1995

Stimulation of cyclic GMP accumulation by sodium nitroprusside is potentiated via a Gs mechanism in intact pinealocytes.

Cyclic GMP accumulation in pinealocytes is elevated > 100-fold by norepinephrine (NE) through a mechanism involving conjoint activation of alpha 1- and beta 1-adrenergic receptors. Little or no stimulation occurs if either alpha 1- or beta 1-adrenergic receptors alone are activated. It appears that alpha 1-adrenergic effects are mediated by Ca2+ acting in part through nitric oxide (NO), and beta 1-adrenergic effects are mediated by Gs. In the study presented here we investigated effects of adrenergic agonists or related postreceptor-active agents on stimulation of pineal cyclic GMP accumulation by the NO generator sodium nitroprusside (NP). The cyclic GMP response to NP (1 mM) was potentiated by NE and isoproterenol (ISO) but not by phenylephrine, indicating that activation of beta 1-adrenergic receptors potentiates the effects of NP. Similarly, vasoactive intestinal peptide (VIP), cholera toxin (CTX), and forskolin, all of which are known to mimic the effects of ISO in this system, also potentiated the effects of NP. In contrast, neither dibutyryl cyclic AMP nor agents that elevate intracellular Ca2+ levels caused marked potentiation of the effects of NP on pineal cyclic GMP. Depletion (90%) of Gs alpha by 21-h treatment with CTX reduced beta-adrenergic potentiation of NP. These findings indicate that beta-adrenergic agonists and VIP potentiate the effects of NP through a mechanism involving Gs. The molecular basis of this action may be an increase in guanylyl cyclase responsiveness to NO.

Adrenergic alpha-Agonists↗

PubMed1993

Developmental appearance of pineal adrenergic-->guanosine 3',5'-monophosphate response is determined by a process down-stream from elevation of intracellular Ca2+: possible involvement of a diffusible factor.

Adrenergic stimulation of the adult pineal gland increases cAMP and cGMP production by over 100-fold. beta-Adrenergic stimulation results in Gs alpha-mediated cyclase activation; alpha 1-adrenergic activation potentiates the beta-adrenergic effects through mechanisms mediated by the intracellular Ca2+ concentration ([Ca2+]i) and Ca(2+)-phospholipid-dependent protein kinase. Development analysis of these responses has indicated that the adrenergic stimulation of cAMP is present several days after birth, but the cGMP response develops only after the second week of life. In the study presented here, the adrenergic-->cGMP response was analyzed in pineal glands from 10- and 25-day-old rats, with the intention of determining the basis of the developmental appearance of this response. Organ culture and tissue homogenate studies indicated that guanylate cyclase activity, cGMP phosphodiesterase activity, and adrenergic elevation of phospholipase-A2 were similar in pineal glands from 10- and 25-day-old rats. Norepinephrine stimulated an increase in [Ca2+]i in dispersed pinealocytes from 10-day-old rats, as has been previously demonstrated in adult pinealocytes. In contrast, several treatments that elevate [Ca2+]i had no effect on cGMP accumulation in forskolin-treated or beta-adrenergically activated glands from 10-day-old rats, but were fully effective in similarly treated glands from 25-day-old rats. However, glands from 10-day-old animals showed a 33-fold accumulation of cGMP when they were cultured together with glands from 25-day-old rats. These studies indicate that whereas many elements in the system that mediate adrenergic regulation of pineal cGMP are fully developed at 10 days of age, the developmental appearance of the cGMP response is triggered by the development of a process down-stream of the alpha 1-adrenergic stimulation of [Ca2+]i, and this process may involve a diffusible factor.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Recoverin in pineal organs and retinae of various vertebrate species including man.

Recoverin is a recently discovered 26 kDa calcium-binding protein, which activates guanylate cyclase in retinal photoreceptors when the intracellular concentration of free calcium drops upon photoexcitation. In this study we examined the distribution of recoverin in retinae and pineal organs of Xenopus laevis larvae, 1-day-old chicken, adult pigeon, albino rat, sheep and man by means of immunocytochemistry. Recoverin immunoreaction was found in all species investigated except for the chicken. In the retina, recoverin immunoreaction was restricted to photoreceptors; all other cell types were immunonegative. In the pineal organ, the recoverin immunoreaction labeled 'pinealocytes of the sensory line', i.e. classical pineal photoreceptors of Xenopus laevis larvae, modified pineal photoreceptors of pigeon, and pinealocytes of mammals. The number of recoverin immunoreactive pinealocytes varied considerably among species of mammals: very few cells were stained in the rat pineal organ, whereas in rabbit, sheep and man, numerous pinealocytes were found to be recoverin-immunoreactive. No immunocytochemical staining was observed after preabsorption of the recoverin antibody with the recombinant protein. Immunoblotting experiments showed that the immunoreaction is due to a protein of 26 kDa in both retina and pineal tissue. Thus, recoverin appears to belong to the family of proteins which are expressed in both retina and pineal organ and are highly conserved in the course of phylogeny. Recoverin may be involved in phototransduction in the directly light-sensitive pineal organs of poikilothermic vertebrates and birds. However, the functional role of recoverin in the mammalian pineal organ, which is not photosensitive, remains unknown.

Animals↗

PubMed1992

Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.

Amino Acid Sequence↗