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FMRI reveals differential activation in the ventral vision pathway during the perception of objects
S25 LONG-CHAIN ACYL CoA’S AND FREE FATTY ACIDS IN RAT BRAIN AFTER HEAD INJURY H. Heller1, J. Deutsch2, A.D. Purdon3, S.I. Rapoport3, M. Horowitz4, E. Shohami1 Depts. of Pharmacology1, Pharmaceutical Chemistry2 and Physiology4, the Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel and 3Laboratory of Neurosciences, National Institute on Aging, NIH, Bethesda, MD, USA Brain injury induces release of endogenous mediators such as excitatory amino acids, cytokines, reactive oxygen species, and the accumulation of free calcium within the cells. Intracellular calcium activates phospholipase C and phospholipase A2 which hydrolyze membrane phospholipids to release free fatty acids (FFA) to the cytosol. Oxidation of arachidonic acid (AA) results in the production of eicosanoids, which mediate an inflammatory response. Acyl-synthetase binds FFA to CoA to form acyl-CoA, whereas acyl-transferase incorporates the acyl group back into the membrane. The loss of membrane phospholipids disturbs the delicate balance between membrane proteins and lipids, and there is evidence that phospholipids metabolism plays a role in the development of secondary injury after trauma. To assess this notion, we compared the temporal changes in the levels of acyl-CoA (using HPLC) and FFA (using GC) in rat brain extracts after closed head injury (CHI), in normothermic and in heat acclimated (ACC, 30 days at 34°C) rats. The latter were previously shown to have facilitated recovery after CHI and cardiac ischemia. ACC rats did not show any changes in basal acyl-CoA level, whereas 20–50% increase in FFA was measured in these rats. 1h after CHI, 2-fold increase in acyl-CoA level was found in normothermic rats only, with resumption to control levels 24 h later. FFA concentration was dramatically decreased (by 90%) at 24 h post CHI in ACC rats only. These results suggest that resistance to trauma and ischemia may be associated with altered phospholipid metabolism, which is among the pathways affected by heat acclimation. DEPOLARIZATION-INDUCED polyADP-RIBOSYLATION OF NUCLEAR PROTEINS IN BRAIN-CORTEX NEURONS: VOLTAGE-DEPENDENT REGULATION OF DNATRANSCRIPTION AND REPAIR S. Hombourg, G. de Murcia*, N. Moran+, E. Priel.#, and M. CohenArmon Cardiac Research Institute, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel; * Lab. of Molecular and Structural Biology, Louis Pasteur University, Strasbourg, France; + Dept. of Brain Research, Weizmann Institute of Science, Rehovot, Israel; # Dept. of Immunology, Ben-Gurion University of the Negev, Beer-Sheva, Israel We have detected a novel signaling mechanism which induces polyADP-ribosylation of nuclear proteins in response to membrane depolarization in neurons of rat brain-cortex. Membrane depolarization was achieved by electrical stimulation or in response to an increased extracellular [K+], and was measured by patch-clamp whole-cell technique in the current-clamp mode. The voltage-induced effect was attributable to a depolarization induced instantaneous activation of polyADP-ribose-polymerase (PARP), which could be reversed by membrane repolarization. This voltage-induced effect is dependent on [Ca+2] in the cytoplasm as well as on glutathione dependent redox potential. Although PARP does not act as a substrate for pertussis-toxin ADP-ribose-transferase, PTX-catalyzed ADP-ribosylation of proteins in the nucleus stimulated polyADP-ribosylation of nuclear proteins. Since polyADP-ribosylation of nuclear proteins modulates their activity, the effect of membrane depolarization on the activity of nuclear proteins that act as a substrate for polyADP-ribosylation, was examined. Depolarization-induced modulation of topoisomerase-I activity has been observed concomitant with activation of PARP, indicating modulation of the activity of nuclear proteins by membrane depolarization. This
evidence may indicate a novel signaling mechanism in neurons, which subjects DNA-transcription and repair to their excitability. MUSCARINIC ACH RECEPTOR INTERACTS WITH THE EXOCYTIC MACHINERY IN A VOLTAGE-DEPENDENT MANNER N. Ilouz, H. Parnas* and M. Linial Department of Biological Chemistry, *Department of Neurobiology, The Otto Loewi Minerva Center for Molecular and Cellular Neurobiology, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The released neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage-dependent. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the SNARE complex. Using rat synaptosomes we show, for the first time, that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, SNAP-25, VAMP and synaptotagmin as shown using both cross-linking and immunoprecipitation. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. This voltage-dependent interaction of mAChRs with the secretory core complex appears most suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals. REPETITION BLINDNESS IN A REDUCED MEMORY LOAD RSVP TASK USING BASIC FEATURES Z. Inbar and S. Hochstein Institute of Life Science and Center for Neural Computation, The Hebrew University of Jerusalem, Jerusalem, Israel Repetition Blindness (RB) is the reduced ability to report the second appearance of an item in a Rapid Serial Visual Presentation (RSVP). The first occurrence does not facilitate the detection of the second, as in the well known Repetition Priming effect, but causes the opposite. The majority of RB paradigms employ long sequences of stimuli with a wide variety of possible responses. Most of them also require maintaining many items in memory during the RSVP (e.g. Park and Kanwisher, 1994). This may confound memory load effects with the primary causes of RB. On the other hand, RB effects achieved by only two stimuli, (e.g. Luo and Caramazza, 1995), do not investigate the influence of the intervening items. In our experiment we investigate RB in a long sequence RSVP task with reduced memory and reporting loads. The perceptual processing of each item in the sequence is also reduced. In each trial, a sequence of 13 different lines is presented after the trial’s ‘target line’. The task is to determine whether the target appears in the sequence once, twice or not at all. RB is seen in reporting ‘one’ in trials with two instances of the target. The stimuli consist of differently colored and oriented lines. The target line is defined by either one or both of these basic dimensions. The RB effect appears to be a function of the gap between the two instances of the target in the sequence. The effect decreases with larger gaps: more items to process between the targets, and of course more time. We study the influence of these parameters on RB.
FMRI REVEALS DIFFERENTIAL ACTIVATION IN THE VENTRAL VISION PATHWAY DURING THE PERCEPTION OF OBJECTS
S26 A. Ishai, L.G. Ungerleider, A. Martin, J.V. Haxby Laboratory of Brain and Cognition, NIMH, Bethesda, Maryland, USA
phorylation of the channel, it regulates the extent of inactivation conferred by the E subunit.
Recently we have demonstrated a dissociation in human ventral extrastriate cortex between subregions that respond more to photographs of faces, houses or chairs (Ishai et al., NeuroImage 1997, 5(4)). In both passive viewing and delayed match-to-sample tasks, responses to faces, measured with functional magnetic resonance imaging (fMRI), were strongest in a subregion in between a more medial subregion that responded more strongly to houses and a more lateral subregion that responded more strongly to chairs. To rule out the possibility that this segregation was due to the surface qualities of the visual stimuli, we have repeated the experiment but with line drawings of faces, houses, and chairs. Changes in blood oxygen level dependent T2*-weighted MRI signal were measured while 6 subjects performed delayed matching tasks with line drawings and photographs. In each time series, 91 volumes, each containing 18 5-mm thick coronal slices, were obtained using gradient echo echoplanar imaging (TR = 3 s, TE = 40 ms, FOV = 20 cm). Data were analyzed using multiple regression to identify regions that responded more to meaningful stimuli as compared to control stimuli, and to test whether these regions responded differentially to faces, houses, and chairs. Visual presentation of both line drawings and photographs of objects activated, bilaterally, the same regions in the fusiform gyrus; an area most responsive to faces was embedded between a medial area most responsive to houses and a lateral area most responsive to chairs. These results indicate that the differential activation in ventral extrastriate cortex cannot be attributed to different surface qualities of the stimuli, but rather are due to some other structural or semantic properties that distinguish these categories of objects.
THE SPLIT CIRCUIT SCHEME: AN ACCOUNT FOR THE SYMPTOMATOLOGY OF HUNTINGTON’S DISEASE D. Joel and I. Weiner Dept. of Psychology, Tel Aviv University, Tel Aviv, Israel
INACTIVATION OF A VOLTAGE-DEPENDENT K+ CHANNEL BY E SUBUNIT: MODULATION BY A PHOSPHORYLATIONDEPENDENT INTERACTION BETWEEN THE DISTAL CTERMINUS OF $ SUBUNIT AND CYTOSKELETON J. Jing, T. Peretz, D. Singer-Lahat, D. Chikvashvili, W.B. Thornhill* and I. Lotan Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel; *Department of Physiology and Biophysics, Mount Sinai School of Medicine, The Mount Sinai Hospital, New York, NY, USA Kv1.1/KvE1.1 (DE) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component in the total current (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the D subunit at Ser446, which impairs the interaction of the channel with microfilaments (Levin et al., (1996) J. Biol. Chem. 271, 29321–29328). The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of D, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C-terminus of D. Truncation of the last six amino acids or mutation of a critical threonine in the TDV sequence comprising the last 3 amino acids, resulted in DE channels with an extent of inactivation up to 2.5fold larger and its further enhancement by cytochalasin being reduced 2-fold. The wild-type (WT) channels exhibited strong inactivation which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the WT channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of membrane-associated synaptic proteins (Kim et al. (1995) Nature 378, 85–88; Kim et al. (1996) Neuropharmacology 35, 993–1000.), we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phos-
The ‘split circuit’ model of basal ganglia-thalamocortical circuitry posits that interaction between basal ganglia-thalamocortical circuits is inherent in the neural architecture of these circuits, so that the same set of connections subserves both segregated and integrated processing. On the basis of the available anatomical data we tentatively identified a motor, an associative, and a limbic split circuit, each containing a closed circuit and an open pathway, terminating in a frontocortical area which is a source of a different circuit. There is a closed indirect pathway (connecting functionally corresponding subregions of the striatum, pallidum, and subthalamic nucleus) within each of the closed circuits as well as within the open associative pathway. In addition, there is an open indirect pathway, connecting the associative striatum to the motor split circuit, and there may be an additional open indirect pathway connecting the associative striatum to the limbic split circuit (Joel and Weiner, Neurosci. 63: 363–379, 1994; Brain Res. Rev. 23: 62–78, 1997). In this manner split circuits are interconnected via open pathways and open indirect pathways. The split circuit scheme can account for the coexistence of motor, cognitive, and emotional symptoms in the early stages Huntington’s disease (HD) when pathology is restricted to the associative striatum. Based on this scheme we suggested that lesion to the external segment of the globus pallidus may ameliorate some of the symptomatology of HD and may also slow down the progressive striatal degeneration. DIFFERENTIAL RECOGNITION OF D-BUNGAROTOXIN AND OF THE SITE-SPECIFIC MONOCLONAL ANTIBODY 5.5 BY THE NICOTINIC ACETYLCHOLINE RECEPTOR S.G. Kachalsky, B.S. Jensen, D. Barchan and S. Fuchs Department of Immunology, Weizmann Institute of Science, Rehovot, Israel The ligand binding site of the nicotinic acetylcholine receptor (AChR) resides mainly in the receptor’s D-subunit, within a short segment including the two tandem cysteines 192 and 193. Our laboratory has been engaged in analyzing the structural requirements for ligand binding by studying AChRs from animals that are resistant to Dneurotoxins, such as the snake and the mongoose. Two competitive antagonists, D-bungarotoxin (D-BTX) and the anti-site monoclonal antibody 5.5 (mAb 5.5), were employed as reagents to follow binding specificities of AChR and of fragments derived from it. We have previously demonstrated that the resistance of the mongoose AChR to DBTX is determined by four amino acid substitutions at the ligand binding region, including substitutions of two aromatic residues (187 and 189) by nonaromatic ones and of two prolines (194 and 197) by leucine and histidine, respectively. A fragment containing the binding site region of the mongoose AChR did not bind D-BTX whereas it did bind mAb5.5, suggesting that the structural requirements for binding of D-BTX and of mAb5.5 are distinct from each other. For a fine analysis of this distinction we have used site directed mutagenesis at unique positions at the ligand-binding site of the mongoose AChR, and compared the binding activities to D-BTX and to mAb5.5. By using the various mutant fragments we have observed a clear differentiation between the interactions of the fragments with the two antagonists. Some mutations bind D-BTX and do not bind mAb5.5, some other mutations do not bind D-BTX and bind mAb5.5, and there are mutations that bind both reagents partially. Altogether, it is concluded from our study that amino acid composition seems more crucial in determining D-BTX binding, whereas conformational parameters are important for recognition of the site-specific mAb5.5.
evidence may indicate a novel signaling mechanism in neurons, which subjects DNA-transcription and repair to their excitability. MUSCARINIC ACH RECEPTOR INTERACTS WITH THE EXOCYTIC MACHINERY IN A VOLTAGE-DEPENDENT MANNER N. Ilouz, H. Parnas* and M. Linial Department of Biological Chemistry, *Department of Neurobiology, The Otto Loewi Minerva Center for Molecular and Cellular Neurobiology, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The released neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage-dependent. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the SNARE complex. Using rat synaptosomes we show, for the first time, that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, SNAP-25, VAMP and synaptotagmin as shown using both cross-linking and immunoprecipitation. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. This voltage-dependent interaction of mAChRs with the secretory core complex appears most suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals. REPETITION BLINDNESS IN A REDUCED MEMORY LOAD RSVP TASK USING BASIC FEATURES Z. Inbar and S. Hochstein Institute of Life Science and Center for Neural Computation, The Hebrew University of Jerusalem, Jerusalem, Israel Repetition Blindness (RB) is the reduced ability to report the second appearance of an item in a Rapid Serial Visual Presentation (RSVP). The first occurrence does not facilitate the detection of the second, as in the well known Repetition Priming effect, but causes the opposite. The majority of RB paradigms employ long sequences of stimuli with a wide variety of possible responses. Most of them also require maintaining many items in memory during the RSVP (e.g. Park and Kanwisher, 1994). This may confound memory load effects with the primary causes of RB. On the other hand, RB effects achieved by only two stimuli, (e.g. Luo and Caramazza, 1995), do not investigate the influence of the intervening items. In our experiment we investigate RB in a long sequence RSVP task with reduced memory and reporting loads. The perceptual processing of each item in the sequence is also reduced. In each trial, a sequence of 13 different lines is presented after the trial’s ‘target line’. The task is to determine whether the target appears in the sequence once, twice or not at all. RB is seen in reporting ‘one’ in trials with two instances of the target. The stimuli consist of differently colored and oriented lines. The target line is defined by either one or both of these basic dimensions. The RB effect appears to be a function of the gap between the two instances of the target in the sequence. The effect decreases with larger gaps: more items to process between the targets, and of course more time. We study the influence of these parameters on RB.
FMRI REVEALS DIFFERENTIAL ACTIVATION IN THE VENTRAL VISION PATHWAY DURING THE PERCEPTION OF OBJECTS
S26 A. Ishai, L.G. Ungerleider, A. Martin, J.V. Haxby Laboratory of Brain and Cognition, NIMH, Bethesda, Maryland, USA
phorylation of the channel, it regulates the extent of inactivation conferred by the E subunit.
Recently we have demonstrated a dissociation in human ventral extrastriate cortex between subregions that respond more to photographs of faces, houses or chairs (Ishai et al., NeuroImage 1997, 5(4)). In both passive viewing and delayed match-to-sample tasks, responses to faces, measured with functional magnetic resonance imaging (fMRI), were strongest in a subregion in between a more medial subregion that responded more strongly to houses and a more lateral subregion that responded more strongly to chairs. To rule out the possibility that this segregation was due to the surface qualities of the visual stimuli, we have repeated the experiment but with line drawings of faces, houses, and chairs. Changes in blood oxygen level dependent T2*-weighted MRI signal were measured while 6 subjects performed delayed matching tasks with line drawings and photographs. In each time series, 91 volumes, each containing 18 5-mm thick coronal slices, were obtained using gradient echo echoplanar imaging (TR = 3 s, TE = 40 ms, FOV = 20 cm). Data were analyzed using multiple regression to identify regions that responded more to meaningful stimuli as compared to control stimuli, and to test whether these regions responded differentially to faces, houses, and chairs. Visual presentation of both line drawings and photographs of objects activated, bilaterally, the same regions in the fusiform gyrus; an area most responsive to faces was embedded between a medial area most responsive to houses and a lateral area most responsive to chairs. These results indicate that the differential activation in ventral extrastriate cortex cannot be attributed to different surface qualities of the stimuli, but rather are due to some other structural or semantic properties that distinguish these categories of objects.
THE SPLIT CIRCUIT SCHEME: AN ACCOUNT FOR THE SYMPTOMATOLOGY OF HUNTINGTON’S DISEASE D. Joel and I. Weiner Dept. of Psychology, Tel Aviv University, Tel Aviv, Israel
INACTIVATION OF A VOLTAGE-DEPENDENT K+ CHANNEL BY E SUBUNIT: MODULATION BY A PHOSPHORYLATIONDEPENDENT INTERACTION BETWEEN THE DISTAL CTERMINUS OF $ SUBUNIT AND CYTOSKELETON J. Jing, T. Peretz, D. Singer-Lahat, D. Chikvashvili, W.B. Thornhill* and I. Lotan Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel; *Department of Physiology and Biophysics, Mount Sinai School of Medicine, The Mount Sinai Hospital, New York, NY, USA Kv1.1/KvE1.1 (DE) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component in the total current (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the D subunit at Ser446, which impairs the interaction of the channel with microfilaments (Levin et al., (1996) J. Biol. Chem. 271, 29321–29328). The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of D, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C-terminus of D. Truncation of the last six amino acids or mutation of a critical threonine in the TDV sequence comprising the last 3 amino acids, resulted in DE channels with an extent of inactivation up to 2.5fold larger and its further enhancement by cytochalasin being reduced 2-fold. The wild-type (WT) channels exhibited strong inactivation which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the WT channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of membrane-associated synaptic proteins (Kim et al. (1995) Nature 378, 85–88; Kim et al. (1996) Neuropharmacology 35, 993–1000.), we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phos-
The ‘split circuit’ model of basal ganglia-thalamocortical circuitry posits that interaction between basal ganglia-thalamocortical circuits is inherent in the neural architecture of these circuits, so that the same set of connections subserves both segregated and integrated processing. On the basis of the available anatomical data we tentatively identified a motor, an associative, and a limbic split circuit, each containing a closed circuit and an open pathway, terminating in a frontocortical area which is a source of a different circuit. There is a closed indirect pathway (connecting functionally corresponding subregions of the striatum, pallidum, and subthalamic nucleus) within each of the closed circuits as well as within the open associative pathway. In addition, there is an open indirect pathway, connecting the associative striatum to the motor split circuit, and there may be an additional open indirect pathway connecting the associative striatum to the limbic split circuit (Joel and Weiner, Neurosci. 63: 363–379, 1994; Brain Res. Rev. 23: 62–78, 1997). In this manner split circuits are interconnected via open pathways and open indirect pathways. The split circuit scheme can account for the coexistence of motor, cognitive, and emotional symptoms in the early stages Huntington’s disease (HD) when pathology is restricted to the associative striatum. Based on this scheme we suggested that lesion to the external segment of the globus pallidus may ameliorate some of the symptomatology of HD and may also slow down the progressive striatal degeneration. DIFFERENTIAL RECOGNITION OF D-BUNGAROTOXIN AND OF THE SITE-SPECIFIC MONOCLONAL ANTIBODY 5.5 BY THE NICOTINIC ACETYLCHOLINE RECEPTOR S.G. Kachalsky, B.S. Jensen, D. Barchan and S. Fuchs Department of Immunology, Weizmann Institute of Science, Rehovot, Israel The ligand binding site of the nicotinic acetylcholine receptor (AChR) resides mainly in the receptor’s D-subunit, within a short segment including the two tandem cysteines 192 and 193. Our laboratory has been engaged in analyzing the structural requirements for ligand binding by studying AChRs from animals that are resistant to Dneurotoxins, such as the snake and the mongoose. Two competitive antagonists, D-bungarotoxin (D-BTX) and the anti-site monoclonal antibody 5.5 (mAb 5.5), were employed as reagents to follow binding specificities of AChR and of fragments derived from it. We have previously demonstrated that the resistance of the mongoose AChR to DBTX is determined by four amino acid substitutions at the ligand binding region, including substitutions of two aromatic residues (187 and 189) by nonaromatic ones and of two prolines (194 and 197) by leucine and histidine, respectively. A fragment containing the binding site region of the mongoose AChR did not bind D-BTX whereas it did bind mAb5.5, suggesting that the structural requirements for binding of D-BTX and of mAb5.5 are distinct from each other. For a fine analysis of this distinction we have used site directed mutagenesis at unique positions at the ligand-binding site of the mongoose AChR, and compared the binding activities to D-BTX and to mAb5.5. By using the various mutant fragments we have observed a clear differentiation between the interactions of the fragments with the two antagonists. Some mutations bind D-BTX and do not bind mAb5.5, some other mutations do not bind D-BTX and bind mAb5.5, and there are mutations that bind both reagents partially. Altogether, it is concluded from our study that amino acid composition seems more crucial in determining D-BTX binding, whereas conformational parameters are important for recognition of the site-specific mAb5.5.