Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains.
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide-binding domains (NBD1 and NBD2) that control its ATP-dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464A) using the patch clamp technique. K464A mutants have an ATP dependence (EC(50) approximate 60 microM) and opening rate at 2.75 mM ATP (approximately 2.1 s(-1)) similar to wild type (EC(50) approximate 97 microM; approximately 2.0 s(-1)). However, K464A's closing rate at 2.75 mM ATP (approximately 3.6 s(-1)) is faster than that of wild type (approximately 2.1 s(-1)), suggesting involvement of NBD1 in nucleotide-dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP's apparent on-rate and acceleration of its apparent off-rate (approximately 2- and approximately 10-fold, respectively). Since the delay of closing by AMP-PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (approximately 0.055 s(-1)) and closing (approximately 0.006 s(-1)) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A-K1250A double mutants close approximately 5-fold faster (approximately 0.029 s(-1)) than K1250A but open with a similar rate (approximately 0.059 s(-1)), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide-dependent closing, which provides important constraints for NBD-mediated gating models.
Powe
Allan C
AC
Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park Drive, Columbia, MO 65211, USA.
Al-Nakkash
Layla
L
Li
Min
M
Hwang
Tzyh-Chang
TC
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D009154:Mutation / Q000235:genetics* / Q000502:physiology*; D009711:Nucleotides / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D014162:Transfection
D007473:Ion Channels; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D008239:Lysine