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Positions of the cytoplasmic end of BK α S0 helix relative to S1-S6 and of β1 TM1 and TM2 relative to S0-S6.

Liu G, Zakharov SI, Yao Y, Marx SO, Karlin A - J. Gen. Physiol. (2015)

Bottom Line: There, in contrast, S0 is closest to the S2-S3 loop, from which position it is displaced on the addition of β1.The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2-S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane.Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Cardiology; Department of Pharmacology; and Department of Biochemistry, Department of Physiology, and Department of Neurology, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Department of Medicine, Division of Cardiology; Department of Pharmacology; and Department of Biochemistry, Department of Physiology, and Department of Neurology, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032.

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Cross-linking TM1 to TM2. (A) Schematic of engineered β1. Mutations G12C and R182C were made in a background of HIS-FLAG-pWT β1 with the additional mutation E13Q. In this construct, the first site susceptible to cleavage by GluC endoproteinase is C-terminal to TM1 at E50. Also, the native E143, just N-terminal to TM2, is the last E in the sequence. There are five native E’s between E50 and E143, but they do not affect the outcome; complete cleavage by GluC results in an N-terminal 7,747-D fragment containing TM1 and a C-terminal 5,406-D fragment containing TM2. Their cross-linking results in a 13,153-D fragment. (B) Immunoblot with anti-FLAG antibody of β1 G12C-R182C after cross-linking with 400 µM QPD in 0.01% saponin and cleavage with GluC (see Materials and methods). Capture and immunoblotting methods were similar to those published previously (Liu et al., 2010; Wu et al., 2013). Cleavage with GluC after cross-linking yielded both an ∼13-kD band and an ∼7.7-kD band. The extent of cross-linking is calculated as (density 13-kD band)/(density 13-kD band + density 7.7-kD band). After DTT reduction, only a 7.7-kD band is seen, showing that GluC cleavage was complete.
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fig4: Cross-linking TM1 to TM2. (A) Schematic of engineered β1. Mutations G12C and R182C were made in a background of HIS-FLAG-pWT β1 with the additional mutation E13Q. In this construct, the first site susceptible to cleavage by GluC endoproteinase is C-terminal to TM1 at E50. Also, the native E143, just N-terminal to TM2, is the last E in the sequence. There are five native E’s between E50 and E143, but they do not affect the outcome; complete cleavage by GluC results in an N-terminal 7,747-D fragment containing TM1 and a C-terminal 5,406-D fragment containing TM2. Their cross-linking results in a 13,153-D fragment. (B) Immunoblot with anti-FLAG antibody of β1 G12C-R182C after cross-linking with 400 µM QPD in 0.01% saponin and cleavage with GluC (see Materials and methods). Capture and immunoblotting methods were similar to those published previously (Liu et al., 2010; Wu et al., 2013). Cleavage with GluC after cross-linking yielded both an ∼13-kD band and an ∼7.7-kD band. The extent of cross-linking is calculated as (density 13-kD band)/(density 13-kD band + density 7.7-kD band). After DTT reduction, only a 7.7-kD band is seen, showing that GluC cleavage was complete.

Mentions: Based on cross-linking, we previously inferred that the extracellular ends of β1 TM1 and TM2 are close (Liu et al., 2010). The intracellular ends are at least close enough for G12C in the TM1 flank and R182C in the TM2 flanks, both four residues from the membrane, to form a disulfide to the extent of 52 ± 4% (n = 3; Fig. 4). The analysis is based on cleavage of β1 with Glu-C at Glu50 and Glu143, which yields an ∼13-kD band if the two flanks are cross-linked and a 7.7-kD band if they are not (see Materials and methods). The β1 extracellular loop is N-glycosylated at Asn80 and Asn142. These residues should be cut out by the Glu-C cleavages. As a control for this, we found that the deglycosylating PNGase F had no effect on the mobilities of the 13- and 7.7-kD fragments (Fig. 4 B).


Positions of the cytoplasmic end of BK α S0 helix relative to S1-S6 and of β1 TM1 and TM2 relative to S0-S6.

Liu G, Zakharov SI, Yao Y, Marx SO, Karlin A - J. Gen. Physiol. (2015)

Cross-linking TM1 to TM2. (A) Schematic of engineered β1. Mutations G12C and R182C were made in a background of HIS-FLAG-pWT β1 with the additional mutation E13Q. In this construct, the first site susceptible to cleavage by GluC endoproteinase is C-terminal to TM1 at E50. Also, the native E143, just N-terminal to TM2, is the last E in the sequence. There are five native E’s between E50 and E143, but they do not affect the outcome; complete cleavage by GluC results in an N-terminal 7,747-D fragment containing TM1 and a C-terminal 5,406-D fragment containing TM2. Their cross-linking results in a 13,153-D fragment. (B) Immunoblot with anti-FLAG antibody of β1 G12C-R182C after cross-linking with 400 µM QPD in 0.01% saponin and cleavage with GluC (see Materials and methods). Capture and immunoblotting methods were similar to those published previously (Liu et al., 2010; Wu et al., 2013). Cleavage with GluC after cross-linking yielded both an ∼13-kD band and an ∼7.7-kD band. The extent of cross-linking is calculated as (density 13-kD band)/(density 13-kD band + density 7.7-kD band). After DTT reduction, only a 7.7-kD band is seen, showing that GluC cleavage was complete.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Cross-linking TM1 to TM2. (A) Schematic of engineered β1. Mutations G12C and R182C were made in a background of HIS-FLAG-pWT β1 with the additional mutation E13Q. In this construct, the first site susceptible to cleavage by GluC endoproteinase is C-terminal to TM1 at E50. Also, the native E143, just N-terminal to TM2, is the last E in the sequence. There are five native E’s between E50 and E143, but they do not affect the outcome; complete cleavage by GluC results in an N-terminal 7,747-D fragment containing TM1 and a C-terminal 5,406-D fragment containing TM2. Their cross-linking results in a 13,153-D fragment. (B) Immunoblot with anti-FLAG antibody of β1 G12C-R182C after cross-linking with 400 µM QPD in 0.01% saponin and cleavage with GluC (see Materials and methods). Capture and immunoblotting methods were similar to those published previously (Liu et al., 2010; Wu et al., 2013). Cleavage with GluC after cross-linking yielded both an ∼13-kD band and an ∼7.7-kD band. The extent of cross-linking is calculated as (density 13-kD band)/(density 13-kD band + density 7.7-kD band). After DTT reduction, only a 7.7-kD band is seen, showing that GluC cleavage was complete.
Mentions: Based on cross-linking, we previously inferred that the extracellular ends of β1 TM1 and TM2 are close (Liu et al., 2010). The intracellular ends are at least close enough for G12C in the TM1 flank and R182C in the TM2 flanks, both four residues from the membrane, to form a disulfide to the extent of 52 ± 4% (n = 3; Fig. 4). The analysis is based on cleavage of β1 with Glu-C at Glu50 and Glu143, which yields an ∼13-kD band if the two flanks are cross-linked and a 7.7-kD band if they are not (see Materials and methods). The β1 extracellular loop is N-glycosylated at Asn80 and Asn142. These residues should be cut out by the Glu-C cleavages. As a control for this, we found that the deglycosylating PNGase F had no effect on the mobilities of the 13- and 7.7-kD fragments (Fig. 4 B).

Bottom Line: There, in contrast, S0 is closest to the S2-S3 loop, from which position it is displaced on the addition of β1.The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2-S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane.Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Cardiology; Department of Pharmacology; and Department of Biochemistry, Department of Physiology, and Department of Neurology, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Department of Medicine, Division of Cardiology; Department of Pharmacology; and Department of Biochemistry, Department of Physiology, and Department of Neurology, Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032.

No MeSH data available.


Related in: MedlinePlus