Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Effects of various mutations at position 79 of mBest2 on the relative permeability and relative conductance of SCN. Serine-79 in mBest2 was mutated into alanine (A), threonine (T), cysteine (C), leucine (L), glutamate (E), arginine (R), or tyrosine (Y) by site-directed mutagenesis. The mutants were separately cotransfected into HEK293 cells with EGFP vectors to mark transfected cells. Single green cells were selected for whole-cell voltage clamp recordings with a high intracellular free [Ca]. The membrane potential was held at 0 mV, stepped to −100 mV for 200 ms, and ramped from −100 mV to 100 mV in 200 ms. Start-to-start interval time was 10 s. Bath solution was either standard solution or one in which 140 mM Cl was replaced with SCN. (A and B) Effect of external SCN on the steady-state current–voltage relationships in S79A (A) and S79Y (B) mutants. I–V relationships obtained after switching to SCN were plotted for representative cells. (C) Average relative permeability ratios (P SCN /P Cl ) of S79 mutants. The relative permeability was calculated from the Goldman-Hodgkin-Katz equation using the difference in reversal potentials with Cl or SCN in the bath. (D) Average relative conductance ratios (G SCN /G Cl ) of SCN and Cl in S79 mutants. Ratios of conductance with SCN or Cl in the bath were obtained from the measurement of the slope of the current–voltage relationship between −25 and +25 mV from reversal potentials. The bars show the means ± SEM with cell number of 8 (S79A), 6 (S79C), 7 (wild type), 10 (S79T), 3 (S79L), 10 (S79Y), 4 (S79R), and 3 (S79E). The reversal potentials and slope conductances were obtained from the first trace after SCN application.

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques: Permeability, Mutagenesis, Transfection, Membrane

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Anion Selectivity and Conductance of Wild-type, S79C, F80C, and F80R mBest2 Currents

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques:

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: SCN slowly blocks conductance in mBest2 S79T mutants. Experiments were performed as indicated in except a 100-ms duration voltage ramp was applied every 2 s. (A) Current–voltage relationships in Cl bath solution and 4 s and 66 s after switching to SCN bath solution. Note that 4 s after changing to SCN solution, the conductance of S79T did not change, but E rev had shifted. After 66 s in SCN, the conductance had decreased to a minimum. (B and D) Time course of the changes in conductance and reversal potentials in S79T (B, n = 5) and wild type (WT) (D, n = 5) after switching the bath to SCN solution. G slope was measured as indicated in . (C) Time constants for SCN effect. G slope after SCN application in B and D was plotted as a function of time. The curves are the best fits to a single exponential. The apparent time constant (τ) is 16.6 s for S79T but 1.7 s for wild type (WT). The time constant for wild type reflects the time required to change the bath.

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques:

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Block of mBest2-S79T–induced currents by extracellular SCN. (A) Current–voltage relationships in S79T-expressing cells with the external solutions in which Cl was replaced with equimolar amounts of [SCN] from 0 to 140 mM as indicated. (B) The I–V curves in A were replotted vs. the driving force (V m –E rev ) for each SCN concentration. (C and D) Dose-dependent block by SCN at +50 mV (C) or −50 mV (D) driving force. The fractional currents at −50 mV (C) or +50 mV (D) driving force for each anion mixture were plotted as a function of [SCN] for wild type (open circles, n = 4) and the S79T mutant (filled circles, n = 6). The data points were fitted to the logistic equation. The wild-type data are from an experiment contemporaneous with the S79T experiment and agree with those we published previously .

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques: Blocking Assay, Expressing, Concentration Assay, Mutagenesis

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Mutations of V78C, F80C, G83C, F84, V86C, T87C in mBest2 change conductance and permeability to SCN. Experimental protocols are indicated in . (A) Relative permeabilities. (B) Relative conductance. Wild type ( n = 10), F80C ( n = 6), V78C ( n = 5), G83C ( n = 3), F84C ( n = 14), V86C ( n = 4), T87C ( n = 6). Conductances and permeabilities were measured immediately after changing the solution to SCN.

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques: Permeability

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Mutations at position 80 change rectification ratios (RR) of mBest2 currents. (A) I–V curves reflecting inward rectification of F80E mutation in Cl or SCN. (B) F80R mutation makes currents outwardly rectifying in Cl or SCN. (C) Rectification ratio of mBest2-wild type (wt, n = 17), F80E ( n = 12), and F80R ( n = 16). The measurement for RR is described in materials and methods .

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques: Mutagenesis

Journal: The Journal of General Physiology

Article Title: Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel

doi: 10.1085/jgp.200409108

Figure Lengend Snippet: Voltage-dependent block of F80R currents by extracellular DIDS. (A) Effect of DIDS on currents induced in HEK cells expressing wild type ( n = 8), F80E mutant ( n = 7), and F80R mutant ( n = 7) mBest2. DIDS (100 μM) was applied to the bath and percent inhibition of currents at +100 mV (open bars) and −100 mV (hatched bars) was measured. (B) Block of wild-type current by DIDS is not voltage dependent. The I–V curves show the voltage-independent block by various [DIDS] in bath solution as indicated. (C) I–V curves showing voltage-dependent block by DIDS of F80R currents. (D) Analysis of voltage-dependent block of F80R currents. Each curve from +10 to +100 mV in C was divided by the curve obtained in 0 μM DIDS. The fractional currents were plotted versus membrane potential. (E) [DIDS]-dependent block of F80R currents. The fractional currents at various potentials in D were replotted as function of [DIDS]. The data were fitted to logistic equation. (F) Voltage dependence of K i of DIDS block. Apparent K i at each voltage was determined from the fits in E in three separate experiments and averaged. The averaged K i was plotted as a function of membrane potential. The points were fitted to Woodhull equation (see text for further details).

Article Snippet: Site-specific mutations of mouse bestrophin-2 (mBest2) were made using a PCR-based site-directed mutagenesis kit (Quickchange; Stratagene) as described previously ( ). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells.

Techniques: Blocking Assay, Expressing, Mutagenesis, Inhibition, Membrane