External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

Area of Science:

• Molecular Biology
• Biophysics
• Plant Physiology

Background:

• The cyclic nucleotide binding domain (CNBD) superfamily includes diverse cation channels, such as depolarization-gated EAG K+ channels and hyperpolarization-gated KAT1 channels.
• In both channel types, the S4 helix of the voltage sensor domain (VSD) moves upon depolarization, but its role in hyperpolarization-gated channels is less understood.
• Acidic charges in the VSD of EAG channels bind divalent cations and protons, inhibiting channel activation.

Purpose of the Study:

• To investigate the effects of external divalent cations (Cd2+) and protons on the plant hyperpolarization-gated channel KAT1.
• To determine if KAT1 shares a similar ion/proton binding pocket with EAG family channels.
• To elucidate the structural basis for KAT1 regulation by Cd2+ and pH.

Main Methods:

• Expression of Arabidopsis thaliana KAT1 in Xenopus oocytes.
• Electrophysiological recordings to measure voltage-dependent activation.
• Site-directed mutagenesis of conserved acidic residues in the KAT1 VSD (D95N, N99D, Q149E).
• Assessment of Cd2+ and proton effects on channel gating.

Main Results:

• External Cd2+ and protons strongly potentiate voltage activation of KAT1.
• Cd2+ shifts the V50 by 150 mV, and acidification (pH 7.0 to 4.0) shifts it by 49 mV.
• Mutating a conserved acidic residue (D95N) eliminated Cd2+ and pH sensitivity.
• Introducing acidic residues (N99D, Q149E) decreased Cd2+ sensitivity and increased proton potentiation.

Conclusions:

• KAT1 is potentiated by external Cd2+ and protons, indicating a distinct regulatory mechanism compared to EAG channels.
• Cd2+ binding is state-dependent, suggesting interaction with an S4-down VSD conformation.
• Plant hyperpolarization-gated channels likely possess a homologous divalent/proton binding site to EAG K+ channels, suggesting conserved gating mechanisms within the CNBD superfamily.