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Biophys J, April 2000, p. 1862-1871, Vol. 78, No. 4

Mutation in Pore Domain Uncovers Cation- and Voltage-Sensitive Recovery from Inactivation in KAT1 Channel

Anna Moroni,* Sabrina Gazzarrini,* Raffaella Cerana,dagger Roberta Colombo,* Jens-Uwe Sutter,Dagger Dario DiFrancesco,§ Dietrich Gradmann,Dagger and Gerhard ThielDagger

 *Dipartimento di Biologia, Università degli Studi and Centro CNR per la Biologia Cellulare e Molecolare delle Piante, Milan, Italy;  dagger DISAT, Università di Milano Bicocca, Milan, Italy;  Dagger A. v. H. Plant Physiology Institute, University of Göttingen, Göttingen, Germany; and  §Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Milan, Italy

Effects of threonine substitution by glutamine at position 256 in the pore of the KAT1 channel have been investigated by voltage-clamp, using heterologous gene expression in Xenopus oocytes. The major discrepancy in T256Q from the wild-type channel (wt) was cation specific. While K+ currents were reduced in a largely scalar fashion, the NH4+ current exhibited slow, voltage-dependent inhibition during hyperpolarization. The same effects could be induced in wt, or intensified in T256Q, by addition of the impermeant cation methylammonium (MA+) to the bath. This stresses that both the mutation and MA+ affect a mechanism already present in the wt. Assuming that current inhibition could be described as entry of the channel into an inactive state, we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme, where C (closed) and I (inactivated) are nonconductive states, and O is an open state allowing K+ and NH4+ passage. The key reaction is the transition I-O. This cation-sensitive transition step ensures release of the channel from the inactive state and is ~30 times smaller in T256Q compared to wt. It can be inhibited by external MA+ and is stimulated strongly by K+ and weakly by NH4+. This sensitivity of gating to external cations may prevent K+ leakage from cation-starved cells.

Biophys J, April 2000, p. 1862-1871, Vol. 78, No. 4
© 2000 by the Biophysical Society   0006-3495/00/04/1862/10  $2.00


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