Purification and Mass Analysis of IbTx-LC-biotin. (A) Reverse phase HPLC chromatogram of crude oxidized (top trace) and purified (bottom trace) synthetic IbTx- LC-biotin. Samples were run on a Vydac C18 column and eluted with a linear gradient of Buffer A to buffer B (A = 0.1% TFA, B = 60% CH₃CN in 0.08% TFA, over 60 min.). (B) Electrospray MS analysis of pure oxidized IbTx-LC-biotin demonstrating a deconvoluted average mass of 4584.1 Da.

Electrophysiological Assay of IbTx-LC-biotin and ChTx on Whole-cell Current of HSlo Expressed in HEK293 Cells. (A) Effect of IbTx-LC-biotin and paxilline on whole-cell currents. Top panel shows a family of current traces elicited by stepping membrane voltage from a holding potential of −80 mV up to +100 mV in consecutive steps of +10 mV for a duration of 100 ms. Middle and bottom panels were recorded after addition of 100 nM IbTx-LC-biotin and 1 μM paxilline, respectively, to the extracellular bathing solution. (B) Plot of whole-cell current from panel A at the end of the 100 ms depolarization vs. the step voltage. Solid and dotted lines for Control and 100 nM IbTx-LC-biotin correspond to current values before and after subtraction of background current measured in the presence of 1 μM paxilline, respectively. (C) Time course of inhibition by toxins and recovery after perfusion. Peak current was monitored by 50 ms voltage steps from −80 mV to +50 mV once every 10 s. IbTx-LC-biotin (200 nM) or ChTx (100 nM) was added to the extracellular perfusion solution at the time of the first arrow and replaced by toxin-free solution at the time of the second arrow. Data points are normalized to the first few points before addition of toxin. Solid lines overlays are fits to an exponential time course. (D) Titration curves of toxin inhibition. HSlo current at steady-state in the presence of various concentrations of IbTx-LC-biotin or ChTx is normalized to values measured before toxin addition. Data is fit to a single-site inhibition isotherm (I_Norm = K_D/(K_D + [toxin]) with K_D values of 26 nM for IbTx-LC-biotin and 13 nM for ChTx.

Stoichiometry of IbTx-LC-biotin Binding to StrAv. (A) Fluorescence titration of 11 pmol StrAv with biotin-4-fluorescein. Open circles, StrAv alone; open squares, StrAv preincubated with 33 pmol IbTx-LC-biotin. The arrow indicates the equivalence point at ∼44 pmol biotin-4-fluorescein. (B) Titration of IbTx-LC-biotin with increasing StrAv. Peak areas corresponding to free IbTx-LC-biotin for various samples in Fig. 4 were normalized to the control taken in the absence of StrAv and plotted versus the amount of added StrAv. Titration data are consistent with an equivalence point near 125 pmol StrAv or a molar ratio of 1 : 0.5 for IbTx-LC-biotin /StrAv. Dashed line shows theoretical expectation for four IbTx-LC-biotin binding sites per StrAv. (C) Space-filling molecular models of StrAv complex with biotin on the left (structure 1SWE from Protein Data Bank) and IbTx-LC-biotin on the right at the same scale. Arrows mark the location of two adjacent biotin sites on the front face of the StrAv tetramer.

Specificity of Fluorescence Labeling by IbTx-LC-biotin Assesed by Various Controls. (A, B) Live (unfixed) HEK293/HSlo cells after processing with 50 nM IbTx-LC-biotin and 3 μg/ml Alexa488-StrAv. Control coverslip in B was treated identically to A except that 5 μM IbTx-D19C-Acm was added before exposure to IbTx-LC-biotin. (C, D) HEK293/HSlo cells after processing with 50 nM IbTx-LC-biotin, 3 μg/ml Alexa488-StrAv, and fixation with 2% formaldehyde. Control coverslip in D was treated identically to C except that IbTx-LC-biotin was omitted. (E, F) HEK293/HSlo cells after processing with 50 nM IbTx-LC-biotin, 4 μg/ml Alexa488-antibiotin antibody, and fixation with 2% formaldehyde. Control coverslip in F was treated identically to E except that IbTX-LC-biotin was omitted. Cells were processed for microscopy and viewed with a 40× oil immersion objective as described in Methods. Scale bar = 30 μm.

Design Strategy and Molecular Model of IbTx-LC-biotin. (A) Sequence of synthetic IbTx-LC-biotin showing substitution of native residue D19 with Lys(ε-d-LC-biotin). Asterisks (*) mark eight residues important for toxin binding to BK channels. (B) Chemical structure of the side chain of the biotin derivative showing the ∼15 Å linker between the Lys α-carbon atom and the biotin moiety buried in the complex with streptavidin. (C) Molecular model of IbTx-LC-biotin. Left panel is a side view of a model of the IbTx derivative with the BK channel binding surface at the bottom and the biotin-derivative side chain at the top. A 90° rotation of the latter structure back into the plane of the paper was used to generate the image for right panel, which shows green colored residues (marked * in A) at the toxin-channel interface.

Bilayer Assay of BK_Ca Channel Block by IbTx-LC-biotin in the Absence and Presence of StrAv. (A) Current record of a single BK_Ca channel from rat skeletal muscle under control conditions in the absence of toxin. (B) Record of a BK_Ca channel with 4 nM IbTx-LC-biotin on the external side. (C) Record of a BK_Ca channel in the presence of 4 nM IbTx-LC-biotin plus 16 nM StrAv on the external side. A small triangle in the third line marks where data is spliced from another bilayer recorded under identical conditions. Control conditions: Holding voltage = +30 mV. The solution on both sides of the bilayer was 10 mM Mops-KOH, pH 7.4, 200 mM KCl. The internal solution also contained 200 μM CaCl₂ and the external solution contained 0.2 mM EDTA plus 0.1 mg/ml bovine serum albumin. Arrows to the right indicate zero current level or the closed state of the channel. Low conductance noise superimposed on IbTx-blocked events in Figs. 3B and 3C is due to activity of extraneous, non-BK, channels occasionally observed in planar bilayer recordings.

Analysis of Complex Formation between IbTx-LC-biotin and StrAv by Size Exclusion Chromatography. A fixed amount of IbTx-LC-biotin (250 pmol) was incubated with increasing amounts of StrAv in the range of 0 to 375 pmol and samples were subjected to HPSEC as described in Methods. Overlayed consecutive traces of the absorbance profile are alternated using solid and dashed lines for sake of clarity. The inset shows seven traces underlying the peak corresponding to unbound IbTx-LC-biotin that have been enlarged and vertically displaced. Triangles mark column calibrations: Vo, void volume; Ve, column volume.

Fluorescence Microscopy of Transfected Cells Expressing HSlo Using IbTx-LC-biotin. (A, C) Stably transfected HEK293/HSlo cells. (B, D) Untransfected HEK293 cells. (A, B) Fluorescence images acquired under identical conditions. (C, D) DIC image of the same fields in A and B, respectively. Cells grown on coverslips were processed for microscopy by incubation with 100 nM IbTx-LC-biotin, then washed and incubated with 10 μg/ml Alexa488-StrAv, washed again, fixed in 2% formaldehyde, and viewed using a 100× oil immersion objective as described in Methods. Scale bar = 10 μm.