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Articles by W. B Guggino
Total Records ( 2 ) for W. B Guggino
  S Atwell , C. G Brouillette , K Conners , S Emtage , T Gheyi , W. B Guggino , J Hendle , J. F Hunt , H. A Lewis , F Lu , I. I Protasevich , L. A Rodgers , R Romero , S. R Wasserman , P. C Weber , D Wetmore , F. F Zhang and X. Zhao

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387–646(405–436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-Å resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542–547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  K. M Hoque , O. M Woodward , D. B van Rossum , N. C Zachos , L Chen , G. P.H Leung , W. B Guggino , S. E Guggino and C. M. Tse

Intestinal Cl secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl secretion. FSK-stimulated Cl secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl>Br>I permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl secretion, which is carried by a novel, previously undescribed Cl channel.

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