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Address correspondence to Dr. Ramon Latorre Laboratory of Biophysics and Molecular Physiology Centro de Estudios Científicos Arturo Prat 514 Valdivia, Chile. Fax: (56) 63-234515; email: ramon{at}cecs.cl
Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (
= 318 ± 34 µs at -180 mV) and a slower component (4.5 ± 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible (
10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage–sensing charges of S4 move inward when the KAT1 channels open.
Key Words: KAT1 channels • inward rectifier • gating currents • cysteine accessibility • voltage sensor
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