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Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation.

ABSTRACT: It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.

SUBMITTER: Szanto TG

PROVIDER: S-EPMC8148028 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na(+) into the cell and K(+) out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a "gating current", a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12-13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the "gate" of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K(+) channels, although Na(+) channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned by the water present within the channel. Our own quantum calculations as well as numerous experiments of others are interpreted in terms of this hypothesis. It is also shown that the evidence that supports the motion of the sensor as the gating current can also be consistent with the hypothesis we present.

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Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation.

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