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

Moroni, A., Gazzarrini, S., Cerana, R., Colombo, R., Sutter, J., Difrancesco, D., et al. (2000). Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel. BIOPHYSICAL JOURNAL, 78(4), 1862-1871 [10.1016/S0006-3495(00)76735-X].

Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel

Cerana, R;
2000

Abstract

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
Articolo in rivista - Articolo scientifico
KAT1 channel, pore domain
English
2000
78
4
1862
1871
none
Moroni, A., Gazzarrini, S., Cerana, R., Colombo, R., Sutter, J., Difrancesco, D., et al. (2000). Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel. BIOPHYSICAL JOURNAL, 78(4), 1862-1871 [10.1016/S0006-3495(00)76735-X].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/711
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