Nucleotides provide a voltage-sensitive gate for the rapid anion channel of Arabidopsis hypocotyl cells

The rapid anion channel of Arabidopsis hypocotyl cells is highly voltage-de pendent. At hyperpolarized potentials, the channel is closed, and membrane depolarization is required for channel activation. We have previously shown that channel gating is regulated by intracellular nucleotides. In the pres ent study, we further analyze the channel gating, and we propose a mechanis m to explain its regulation by voltage. In the absence of intracellular nuc leotides, closure at hyperpolarized voltages is abolished. Structure-functi on studies of adenyl nucleotides show that the apparent gating charge of th e current increases with the negative charge carried by nucleotides. We pro pose that the fast anion channel is gated by the voltage-dependent entry of free nucleotides into the pore, leading to a voltage-dependent block at hy perpolarized potentials. In agreement with this mechanism in which intracel lular nucleotides need to be recruited to the channel pore, kinetic analyse s of whole-cell and single-channel currents show that the rate of closure i s faster when intracellular nucleotide concentration is increased, whereas the rate of channel activation is unchanged. Furthermore, decreasing the co ncentration of extracellular chloride enhances the intracellular nucleotide block. This result supports the hypothesis of a mechanism in which blockin g nucleotides and permeant anions interact within the channel pore.