Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SK<sub>Ca</sub> Channels

Wu, Jinfeng; He, Linlin; Zhou, Zhuan; Chi, Cheng-Wu

doi:10.1021/bi011367z

Cited by 35 publications

(32 citation statements)

References 24 publications

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“…Given their abundance in crude venom and variation across species, the ␣D-conotoxins appear to have evolved as an alternative strategy for prey capture for species lacking ␣-, ␣A-, ␣S-, and -conotoxins. Whereas chemical synthesis of these proteins is expected to be challenging, recombinant production of other dimeric toxins of comparable size has been achieved (49,(62)(63)(64)(65)(66)(67). Co-crystallization studies with AChBP are expected to more precisely define the structure of ␣D-conotoxins and to establish the molecular basis of their binding interaction at the nAChR.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Class of Nicotinic Receptor Antagonists

Loughnan

Nicke

Jones

et al. 2006

Journal of Biological Chemistry

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The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, ␣D-VxXIIA, ␣D-VxXIIB, and ␣D-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ϳ11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that ␣D-VxXIIA, ␣D-VxXIIB, and ␣D-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for ␣7 and ␤2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs. Nicotinic acetylcholine receptors (nAChRs)4 belong to the Cys-loop superfamily of pentameric ligand-gated ion channels. Neuronal nAChRs are generally formed from a combination of ␣ and ␤ subunits (␣2-␣10 and ␤2-␤4) that can assemble into a diversity of nAChR subtypes with different pharmacological and functional properties (1). Given their physiological importance, nAChRs are often targeted by venom peptides (2).Our current knowledge of the structure and function of nAChRs owes much to studies using snake toxins as biochemical or pharmacological tools to isolate and characterize this receptor (3). The "bungarotoxin" family of proteins in the venom of elapid and hydrophid snakes includes the -and ␣-neurotoxins, muscarinic toxins, cytotoxins, cardiotoxins, fasciculins, calciseptins, and mambins (4, 5). The structurally related short-chain and long-chain ␣-neurotoxins and the "weak non-conventional" snake toxins are comprised of 60 -74 amino acids, including 8 or 10 cystine residues and their structures have a three-fingered fold. The -and ␣-neurotoxins both inhibit nAChRs but differ in their specificity and binding kinetics (4).Conotoxins are small disulfide-rich peptide toxins found in the venom of predatory marine snails from the genus Conus. These venom peptides generally target a variety of voltage-gated and ligand-gated ion channels (6, 7). Conotoxins acting at nAChRs include the ␣-conotoxin, ␣A-conotoxin, -conotoxin, and ␣S-conotoxin families. The ␣-conotoxins are a large family of well characterized competitive nAChR antagonists with diverse subtype selectivities that allow the pharmacological dissection of nAChR subtypes (1,8). The structurally different ␣A-conotoxins are competitive antagonists with specificity for muscle nAChRs but lack the selectivity for the ␣1/␦ interface that is ex...

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Section: Discussionmentioning

confidence: 99%

Identification of a Novel Class of Nicotinic Receptor Antagonists

Loughnan

Nicke

Jones

et al. 2006

Journal of Biological Chemistry

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“…Adrenal chromaffin cells from Wistar rats (SLACCAS Inc., Shanghai) were isolated and cultured as de-scribed (26,27). Cells were used in the experiments after 2-6 days in culture.…”

Section: Methodsmentioning

confidence: 99%

Calcium influx through hyperpolarization-activated cation channels ( I _h channels) contributes to activity-evoked neuronal secretion

Duan

Shang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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106

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The hyperpolarization-activated cation channels (Ih) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca 2؉ can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and Ih channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca 2؉ imaging, we found that there is a Ca 2؉ influx in proportion to Ih induced by hyperpolarization in HEK293 cells. The Ih channel blockers Cs ؉ and ZD7288 inhibit both HCN current and Ca 2؉ influx. Measurements of the fractional Ca 2؉ current showed that it constitutes 0.60 ؎ 0.02% of the net inward current through HCN4 at ؊120 mV. This fractional current is similar to that of the low Ca 2؉ -permeable AMPA-R (␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of Ih for 30 s also resulted in a Ca 2؉ influx and an elevated action potentialinduced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for Ih channels in modulating neuronal secretion by permitting Ca 2؉ influx at negative membrane potentials.

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“…To compare the effects of BmP09 and BmK AS-1 in the patch clamp experiments, we used rat adrenal chromaffin cells (3,16,19). Dispersion of chromaffin cells was typically done on adrenal medullas from two or three Wistar mice (20 -30 g) ϳ1 month of age.…”

Section: Electrophysiology and Solutionsmentioning

confidence: 99%

BmP09, a “Long Chain” Scorpion Peptide Blocker of BK Channels

Yao

Chen

et al. 2005

Journal of Biological Chemistry

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A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na ؉ channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na ؉ channel blocker to a K ؉ channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca 2؉ -and voltage-dependent K ؉ channels (BK) but not Na ؉ channels at a range of 100 nM concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 ␣-subunits in Xenopus oocytes. The half-maximum concentration EC 50 of BmP09 was 27 nM, and the Hill coefficient was 1.8. In outside-out patches, the 100 nM BmP09 reduced ϳ70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic -interactions around the center.

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Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SK_Ca Channels

Cited by 35 publications

References 24 publications

Identification of a Novel Class of Nicotinic Receptor Antagonists

Identification of a Novel Class of Nicotinic Receptor Antagonists

Calcium influx through hyperpolarization-activated cation channels ( I _h channels) contributes to activity-evoked neuronal secretion

BmP09, a “Long Chain” Scorpion Peptide Blocker of BK Channels

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Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SKCa Channels

Cited by 35 publications

References 24 publications

Identification of a Novel Class of Nicotinic Receptor Antagonists

Identification of a Novel Class of Nicotinic Receptor Antagonists

Calcium influx through hyperpolarization-activated cation channels ( I h channels) contributes to activity-evoked neuronal secretion

BmP09, a “Long Chain” Scorpion Peptide Blocker of BK Channels

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Product

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Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SK_Ca Channels

Calcium influx through hyperpolarization-activated cation channels ( I _h channels) contributes to activity-evoked neuronal secretion