ABCMdb

PMID: 21576373

Szollosi A, Muallem DR, Csanady L, Vergani P
Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.
J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16., [PubMed]

Sentences

No. Mutations Sentence Comment

24 ABCC7 p.Lys1250Arg ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Login to comment 27 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1348Ala Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. Login to comment 36 ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala ABCC7 p.His1348Ala ABCC7 p.His1348Ala M AT E R I A L S A N D M E T H O D S Molecular biology pGEMHE-WT (Chan et al., 2000), carrying the coding sequence of human WT CFTR, was used as a template for mutants T460S, L1353M, H1348A, H1375A, T460S/L1353M, T460S/H1348A, and T460S/H1375A. Login to comment 37 ABCC7 p.Lys1250Arg pGEMHE-K1250R (Vergani et al., 2005) was used as a template for the corresponding nonhydrolytic mutants. Login to comment 59 ABCC7 p.Lys464Ala A mutation in the conserved Walker A lysine of NBD1, K464A, reduced channel opening rate at low [ATP] (Vergani et al., 2003), suggesting that ATP binding at site 1 can be made rate limiting for channel opening. Login to comment 61 ABCC7 p.Lys464Ala In other studies, neither the K464A mutation (Powe et al., 2002) nor mutation of an aromatic residue (W401), which is seen to stack against the adenine moiety of ATP in some crystals of CFTR NBD1 (Lewis et al., 2005, but cf. Login to comment 79 ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser Fig. S2 illustrates experiments to assay the rate of unlocking from the pyrophosphate-induced locked-open state for WT, T460S, L1353M, and T460S/L1353M channels. Login to comment 102 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala Fitting the relaxation time course after ATP removal for the nonhydrolytic H1375A/K1250R and T460S/H1375A/K1250R constructs consistently required a double exponential with two slow time constants (each in the seconds range), suggesting two populations of open-channel bursts (see Fig. 9 A). Login to comment 112 ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser Effects of mutations at positions 460 and 1353 on ATP-dependent (hydrolytic) gating We first tested changes in energetic coupling between positions 460 and 1353 by perturbing these positions using mutations T460S and L1353M. Login to comment 115 ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser To determine if the mutations T460S and L1353M, individually or together, had any effect on channel gating in saturating 2 mM ATP, burst durations were determined from patches containing 1-10 channels (Fig. 2 A). Login to comment 116 ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser There was a small increase in closing rate (defined as inverse of the mean burst duration; Fig. 2 B) for T460S (3.6 ± 0.3 s1 ; n = 20; Fig. 2 B, red bar) and L1353M (3.3 ± 0.4 s1 ; n = 8; Fig. 2 B, blue bar) compared with WT (2.6 ± 0.3 s1 ; n = 13; Fig. 2 B, black bar), whereas there was no significant change for T460S/L1353M (n = 9; Fig. 2 B, green bar). Login to comment 134 ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser We confirmed this was also the case for T460S and L1353M using multichannel analysis on patches containing <10 channels (not depicted), which showed that when [ATP] was reduced from 2 mM to 50 µM, burst duration was not significantly affected, and the fractional Po supported by 50 µM ATP (0.39 ± 0.07 and n = 6 for T460S, and 0.51 ± 0.08 and n = 5 for L1353M) could be accounted for by the fractional opening rate observed under the same conditions (0.39 ± 0.06 and n = 6 for T460S, and 0.46 ± 0.07 and n = 5 for L1353M). Login to comment 151 ABCC7 p.Lys1250Arg In CFTR, the equivalent mutation, K1250R, caused an increase in open burst duration (Vergani et al., 2005; Csanády et al., 2006), consistent with blocking of the fast hydrolytic closure pathway. Login to comment 152 ABCC7 p.Lys1250Arg ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser To determine if the mutations T460S, L1353M, and T460S/L1353M increased the rate of nonhydrolytic closure from an open state with ATP bound at both composite sites, we introduced the above site-1 mutations in a K1250R background. Login to comment 154 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser The fitted time constant for current decay, relaxation (Fig. 5 A, inset), provided an estimate for the average lifetime of the open state, which was 5.9 ± 0.5 s (n = 13) for K1250R (black bar) and unchanged in L1353M/ K1250R (7.2 ± 0.8 s; n = 10; P = 0.11; blue bar), but significantly reduced in T460S/K1250R (4.2 ± 0.3 s; n = 13; P < 0.01; red bar). Login to comment 155 ABCC7 p.Lys1250Arg ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser Because relaxation was additively affected in T460S/L1353M/K1250R (4.5 ± 0.6 s; n = 10; P < 0.05; green bar), G‡ int(closing) was not significantly different from zero (Fig. 5 B), indicating that the coupling between the two residues on opposite sides of composite site 1 was not changed along the nonhydrolytic closure pathway between the ATP-bound open state and the transition state. Login to comment 165 ABCC7 p.Thr460Ser (A) Representative traces showing macroscopic current response for WT and T460S to a test [ATP] of 50 µM, bracketed with applications of 2 mM ATP. Login to comment 172 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala Energetic coupling between positions 460 and 1348 is little changed during gating Following a similar methodology, we proceeded to study changes in coupling between positions 460 and 1348 during gating, using perturbations T460S and H1348A. Login to comment 173 ABCC7 p.Thr460Ser In these mutant cycles, two of the corners (WT and single-mutant T460S) are identical to the corresponding corners of the respective T460-L1353 mutant cycle. Login to comment 174 ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala ABCC7 p.His1348Ala To rigorously compare the effects of the H1348A mutation onto the T460S versus WT backgrounds, the gating parameters for the latter two constructs should have been repeatedly measured in experiments side by side with those conducted on H1348A and T460S/H1348A. Login to comment 175 ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.His1348Ala However, because Gint can be calculated using any two parallel sides of a mutant cycle, we did not repeat experiments for WT and T460S; instead, we calculated Gint using the two horizontal sides of each cycle, i.e., by comparing the effects of the T460S mutation onto the H1348A versus WT backgrounds. For this reason, we refrain from providing absolute G values for the vertical sides of the T460-H1348 mutant cycles (Figs. 6, B and D, and 7, B and D); and the same applies for the T460-H1375 mutant cycles (see below; Figs. 8, B and D, and 9, B and D). Login to comment 176 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala We first studied the single-channel gating pattern of H1348A and T460S/H1348A under normal hydrolytic conditions (Fig. 6 A) and extracted single-channel closing rates (Fig. 6 B, left). Login to comment 177 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala ABCC7 p.His1348Ala Although the H1348A mutation dramatically slowed closure (to 1.1 ± 0.2 s1 ; n = 8), the closing rate for T460S/H1348A was slightly accelerated relative to that of H1348A (compare green and blue bar (Gunderson and Kopito, 1994; Carson et al., 1995; Tsai et al., 2009), likely by inhibiting hydrolytic closure (Scott-Ward et al., 2007; Tsai et al., 2009). Login to comment 178 ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser Therefore, as an alternative means to study nonhydrolytic channel closing rates, we also determined the effect of mutations T460S, L1353M, and T460S/L1353M on the closing of channels locked open by ATP plus PPi (Fig. S2). Login to comment 182 ABCC7 p.Lys1250Arg However, in constructs carrying the K1250R mutation, the hydrolytic pathway is effectively blocked, and the gating cycle, in saturating [ATP], is reduced to a simple equilibrium between the open and closed states. Login to comment 183 ABCC7 p.Lys1250Arg Thus, we can use Po in the K1250R background to determine the free energy difference between the open and closed states for each of the constructs (Gopen-closed) and analyze the results using mutant cycle formalism. Login to comment 185 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Leu1353Met ABCC7 p.Leu1353Met ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser Consistent with changes in closing rate, Po was significantly reduced for T460S/K1250R (0.28 ± 0.06; n = 6; P < 0.01; Fig. 5 C, red bar) and T460S/L1353M/ K1250R (0.26 ± 0.03; n = 8; P < 0.01; green bar) compared with K1250R (0.55 ± 0.07; n = 9; black bar), but not for L1353M/K1250R (0.55 ± 0.05; n = 8; blue bar). Login to comment 186 ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser Here too, the coupling energy, Gint(open-closed), was not significantly different from zero (Fig. 5 D), indicating that there was Figure 5. The T460S mutation destabilizes the open state of CFTR in the nonhydrolytic K1250R background. Login to comment 194 ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1348Ala nonhydrolytic relaxation nor the reduction in nonhydrolytic Po by the T460S mutation (compare red vs. black bars in Fig. 5, A and C) was apparent when this mutation was introduced into an H1348A/K1250R background (compare green vs. blue bars in Fig. 7, A and D, left), these deviations from additivity resulted in a small change in T460-H1348 interaction energy only between the transition state for nonhydrolytic closure and the open ground state (Gint(closing) = 0.43 ± 0.14 kT; P = 0.01; Fig. 7 B), but not between open and closed ground states (Fig. 7 D, right; P = 0.1). Login to comment 195 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala ABCC7 p.His1348Ala Energetic coupling between positions 460 and 1375 is little changed during gating To study interactions between positions 460 and 1375, we compared the effects of mutation T460S in H1375A and WT backgrounds. For single channels gating under normal hydrolytic conditions (Fig. 8 A, top), mutation H1375A also slowed closure (to 1.3 ± 0.1 s1 ; n = 6; Fig. 8 B, left, blue bar), similarly to what was observed for H1348A (see Fig. 6 B, left, blue bar). Login to comment 196 ABCC7 p.Thr460Ser A slight tendency of mutation T460S to accelerate channel closure was also observed in this background (see Fig. 8 B, left, green bar), such that a mutant cycle built on closing rates (Fig. 8 B, right) did not reveal significant nonadditivity (Gint ≈ 0). Login to comment 197 ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala The increased open probability of T460S/H1375A relative to that of H1375A (Fig. 8 C, in Fig. 6 B, left), just as that of T460S relative to WT (compare red and black bar in Fig. 2 B). Login to comment 199 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala The slight difference in closing rates between T460S/H1348A and H1348A was mirrored by the slightly lower Po value of the double mutant (Fig. 6 C; compare green and blue bar). Login to comment 200 ABCC7 p.Thr460Ser Consequently, for the calculated opening rates (Fig. 6 D, left), we did not detect the slight acceleration by the T460S mutation, which was observed when this mutation was introduced into a WT background (compare red and black bars in Fig. 3 C). Login to comment 202 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala To look for changes in interactions between positions 460 and 1348 during nonhydrolytic closure, we created nonhydrolytic H1348A/K1250R and T460S/H1348A/ K1250R channels and compared their closing rates by studying macroscopic current relaxations after ATP removal (Fig. 7 A). Login to comment 203 ABCC7 p.Lys1250Arg ABCC7 p.His1348Ala ABCC7 p.His1348Ala Interestingly, mutation H1348A prolonged the time constant of the current relaxation (to 20 ± 2 s; n = 8; Fig. 7 A, blue bar) to a similar extent as it did normal burst durations; and noise analysis (Fig. 7 C) attested to the fact that the prolonged open time of H1348A/K1250R is associated with an unusually high open probability (Po = 0.83 ± 0.03 s; n = 6; Fig. 7 D, left, blue bar). Login to comment 205 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (A) Representative single-channel current traces from prephosphorylated H1348A and T460S/H1348A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. Login to comment 206 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (B; left) Closing rates of H1348A (blue bar) and T460S/H1348A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). Login to comment 208 ABCC7 p.Thr460Ser The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Login to comment 209 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala Because the bottom two corners (representing H1348A and T460S/H1348A) were evaluated in separate sets of experiments, the absolute G values are not printed for the vertical sides of the cycle. Login to comment 210 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (C) Noise analysis was used to estimate Po for H1348A (blue bar) and T460S/H1348A (green bar). Login to comment 211 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (D; left) Opening rates of H1348A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and the closing rate (see B). Login to comment 214 ABCC7 p.Lys1250Arg We next studied the effect of the mutations at our target pair T460-H1375 on nonhydrolytic closing rate, in a K1250R background. Login to comment 215 ABCC7 p.Lys1250Arg ABCC7 p.His1375Ala Because for both H1375A/K1250R green vs. blue bar) attested to an increased opening rate in the double mutant (Fig. 8 D, left, green vs. blue bar). Login to comment 216 ABCC7 p.Lys1250Arg ABCC7 p.His1348Ala However, because a similar tendency (although to a lesser extent) was also apparent in the WT background (see Fig. 3 C, red vs. black bar), the mutant cycle built Figure 7. The H1348A mutation stabilizes the open state of CFTR in the nonhydrolytic K1250R background. Login to comment 217 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (A) Representative normalized decay time courses of macroscopic currents for H1348A/K1250R and T460S/ H1348A/K1250R CFTR after the removal of 2 mM ATP (gray). Login to comment 221 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (C) Noise analysis for estimation of Po for H1348A (blue symbols) and T460S/H1348A (green symbols); each symbol represents one patch. Login to comment 222 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala (D; left) Mean ± SEM Po for H1348A (blue bar) and T460S/ H1348A (green bar). Login to comment 225 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (A) Representative single-channel current traces from prephosphorylated H1375A and T460S/H1375A CFTR channels gating in 2 mM ATP. Downward deflection indicates inward current. Login to comment 226 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (B; left) Closing rates of H1375A (blue bar) and T460S/H1375A (green bar), defined as the inverse of the mean burst duration (see Materials and methods). Login to comment 228 ABCC7 p.Thr460Ser The top two corners of the mutant cycle (representing WT and T460S) were taken from Fig. 2 C. Login to comment 229 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (C) Noise analysis was used to estimate Po for H1375A (blue bar) and T460S/H1375A (green bar). Login to comment 230 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1348Ala (D; left) Opening rates of H1375A (blue bar) and T460S/H1348A (green bar), obtained using the estimate for Po (see C) and closing rate (see B). Login to comment 239 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn Site-1 mutations affect channel closing, likely by affecting the relative stability of open states In contrast to composite site-2 mutations R555K and T1246N (Vergani et al., 2005, but compare Teem et al., 1996), in positions equivalent to T460 and L1353, all site-1 mutations studied here had relatively small effects on channel gating, consistent with the notion that the gating cycle is driven by the catalytic cycle at composite site 2. Login to comment 240 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala The most significant phenotypes were observed for T460S and H1348A, which, respectively, increased and decreased not only normal hydrolytic channel closing rate (Figs. 2 B and 6 B) but also the rate of nonhydrolytic closure (Figs. 5 A and 7 A; compare Fig. S2 B), suggesting that these mutations simultaneously affect the energy barriers for both closing pathways. Login to comment 241 ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1375Ala An alteration and T460S/H1375A/K1250R adequate fitting of the relaxation time course after ATP removal consistently required a double exponential with two slow time constants (each in the seconds range; Fig. 9 A), average steady-state closing rate was estimated from a double-exponential fit as described in Materials and methods. Login to comment 242 ABCC7 p.Lys1250Arg ABCC7 p.His1375Ala Unexpectedly, when the H1375A mutation was introduced into the K1250R background, average nonhydrolytic closing rate was not slowed, but rather slightly accelerated (Fig. 9 A, blue bar). Login to comment 243 ABCC7 p.Thr460Ser Although in this double-mutant background the T460S mutation did not noticeably affect the rate of nonhydrolytic closure (Fig. 9 A, green bar; compare with Fig. 5 A, red vs. black bar), this small deviation from additivity did not result in any significant coupling energy (Fig. 9 B; P = 0.2). Login to comment 244 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1375Ala Finally, by noise analysis (Fig. 9 C), mutation T460S reduced Po in the H1375A/K1250R background (compare green vs. blue bars in Fig. 9 D, left) to a similar extent as it did in the single-mutant K1250R background (compare red vs. black bars in Fig. 5 C), yielding a Gint(open-closed) not significantly different from zero (Fig. 9 D; P = 0.15). Login to comment 247 ABCC7 p.Lys1250Arg To validate this proposal using an independent approach, we resorted to thermodynamic Figure 9. Effects of mutations at positions 460 and 1375 on nonhydrolytic gating in the K1250R background. Login to comment 248 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (A) Representative normalized decay time courses of macroscopic currents for H1375A/K1250R and T460S/H1375A/K1250R CFTR after the removal of 2 mM ATP. Solid blue and green lines are fitted bi-exponentials. Login to comment 249 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala Fitted parameters were 1 = 2.8 s, 2 = 11 s, A1 = 0.77, and A2 = 0.23 for the H1375A/ K1250R trace, and 1 = 2.8 s, 2 = 15 s, A1 = 0.82, and A2 = 0.18 for the T460S/H1375A/ K1250R trace. Login to comment 253 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (C) Noise analysis for estimation of Po for H1375A (blue symbols) and T460S/H1375A (green symbols); each symbol represents one patch. Login to comment 254 ABCC7 p.Thr460Ser ABCC7 p.His1375Ala ABCC7 p.His1375Ala (D; left) Mean ± SEM Po for H1375A (blue bar) and T460S/H1375A (green bar). Login to comment 274 ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg ABCC7 p.Thr460Ser ABCC7 p.Thr460Ser ABCC7 p.His1348Ala ABCC7 p.His1348Ala Our results on T460S/K1250R and H1348A/K1250R indeed show a respective decrease and increase in Po (Figs. 5 C and 7 D), confirming that the open ground state is destabilized in T460S/K1250R, but stabilized in H1348A/K1250R, with respect to the closed ground state. Login to comment 275 ABCC7 p.Thr460Ser ABCC7 p.His1348Ala The simplest interpretation is that the T460S and H1348A mutations specifically affect the stability of the open state. Login to comment 277 ABCC7 p.His1375Ala In contrast to the simple interpretation of the effects of the above two mutations, the functional consequences of the H1375A mutation are more complex. Login to comment 278 ABCC7 p.Lys1250Arg This mutation affected the rates of hydrolytic (Fig. 8 B) and nonhydrolytic (Fig. 9 A) closure in opposite ways, and in the K1250R background, the double-exponential relaxation after ATP removal suggested a mixture of two types of nonhydrolytic bursts. Login to comment 281 ABCC7 p.His1375Ala Although the H1375A phenotypes are not easily interpretable, altogether our findings are generally consistent with evidence from other studies of mutations in site 1, which suggest that protein-nucleotide and protein-protein interactions at composite site 1 contribute to the stability of the open-state dimer (Vergani et al., 2003; Bompadre et al., 2005; Cai et al., 2006; Zhou et al., 2006; Tsai et al., 2009). Login to comment 289 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. J. Biol. Chem. 281:1970-1977. doi:10 .1074/jbc.M510576200 Carson, M.R., M.C. Winter, S.M. Travis, and M.J. Welsh. 1995. Login to comment