DePuy et al. 10.1073/pnas.0603945103. |
Fig. 5. Neither recombinant Gb2g2 nor Gb1g2 alter a1H channel open times or unitary conductance. (A-B) Open-time histograms plot logarithmically binned open-state durations concatenated from five sets of records (per group) against the square root of events per bin. Open-state dwell-time distributions were best-fit by the sum of two exponentials, and the first (t1) and second (t2) time constants are shown. The percentage of total openings of the channel described by t1 and t2 are indicated in parentheses. (C) Unitary current-voltage relationship. Data were obtained from three to five patches at each test potential by using 60 mM CaCl2 as the charge carrier. The straight line is the linear regression fit of all data, yielding a slope conductance of 6.4 pS (r = 0.95).
Fig. 6. Schematic structural model of Gb2. Locations of amino acids selected for mutation are indicated: Group A, Group B, and residue 140.
Fig. 7. Mutant Gb2- and Gb1-containing dimers retain functionality and stimulate G protein-activated inwardly rectifying K (GIRK)1,4 channels. (A) Sample currents elicited by a voltage ramp protocol (-120 to -50 mV) recorded from HEK-293 cells stably expressing GIRK1,4 channels and coexpressing marker GFP/Gg2 and empty vector or mutant Gb constructs b2A, b2GA, and b1PGA. (B) Histogram compares means ± SEM of slope conductances (pA/mV), calculated from -120 to -100 mV among groups of cells expressing unique Gbg2-dimer combinations. Numbers indicate cells recorded for each condition. Asterisk, P < 0.05 compares K+ conductance of GFP/empty vector-expressing cells with that of Gbxg2 by ANOVA.
Supporting Methods
Molecular Biology.
Point mutations were introduced alone or in sequence by using complementary primers (forward): b2(P140A): 5'-GGG TCA GCC GGG AGC TGG CTG GCC ACA CTG G-3'; b1(A140P): 5'-GTG AGT CGT GAG CTG CCA GGA CAC ACA GGT TAC C-3'; b1GB(Y111F): 5'-CCC TCA GGG AAC TAT GTG GCC TGT GGG-3'; and b1GB(N125S): 5'-CTG CTC CAT CTA CAA CCT CAA GAC-3'. Mutations of GA residues were introduced together by using a megaprimer generated by primer extension with Pfu-Turbo polymerase. b2GA: 5'-GAC ATT GAG ACA GGC CAG CAG ACA ACG ACT TTT ACT GGA CAC AG-3' and 5'-GGA CAT CAC ATC CCC ACT GTG TCC AGT AAA AGT CGT TGT CTG CTG-3'; and b1GA: 5'-CGA GAC CGG CCA GCA GAC GTT CGG GTT TGC CGG ACA CAC TGG AG-3' and 5'-GCT CAT GAC ATC TCC AGT GTG TCC GGC AAA CCC GAC CGT CTG CTG-3'.Single-Channel Analysis.
Averaged blank sweep templates were used to subtract the leak and capacitive currents. Channel openings were detected with a 50% threshold crossing criterion. Open probability (NPo) was calculated as the ratio of the sum of channel open time (per sweep) and the analyzed test pulse duration [Barrett PQ, Lu HK, Colbran R, Czernik A, Pancrazio JJ (2000) Am J Physiol 279:C1694-C1703]. Open-state dwell-time distributions were determined from records, with evidence of few multiple openings (1% or less).Surface-Expression Measurements.
Briefly, cells were rinsed and fixed for 5 min in 4% paraformaldehyde and washed three times for 5 min in PBS, and half of the wells were permeabilized with 0.1% Triton X-100 (5 min) and rinsed three times in PBS. The cells were then incubated for 30 min in blocking solution (PBS plus 1% FBS). The a1H-HA channel expression was detected by using a monoclonal rat anti-HA antibody (1/1,000, clone 3F10; Roche Applied Science, Indianapolis, IN) and incubated for 1 hour at room temperature. After extensive washes (four times for 10 min in PBS plus 1% FBS), cells were incubated for 30 min with the secondary antibody coupled to horseradish peroxidase (1/1,000, The Jackson Laboratory, Bar Harbor, ME). Cells were washed (four times for 10 min in PBS) before the addition of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL). The luminescence was measured by using a Victor 2 luminometer (PerkinElmer, Wellesley, MA), and a BCA assay (Pierce Biotechnology) was then performed to normalize the measurements to the total amount of protein in each well. The finalized data were normalized to the level of signal obtained in the a1H-HA + Gg2 condition (nonpermeabilized) (data were similar to those obtained with a1H-HA channel alone, data not shown).