Two Mechanisms of K+-Dependent Potentiation in Kv2.1 Potassium Channels

Abstract

Elevation of external [K(+)] potentiates outward K(+) current through several voltage-gated K(+) channels. This increase in current magnitude is paradoxical in that it occurs despite a significant decrease in driving force. We have investigated the mechanisms involved in K(+)-dependent current potentiation in the Kv2.1 K(+) channel. With holding potentials of -120 to -150 mV, which completely removed channels from the voltage-sensitive inactivated state, elevation of external [K(+)] up to 10 mM produced a concentration-dependent increase in outward current magnitude. In the absence of inactivation, currents were maximally potentiated by 38%. At more positive holding potentials, which produced steady-state inactivation, K(+)-dependent potentiation was enhanced. The additional K(+)-dependent potentiation (above 38%) at more positive holding potentials was precisely equal to a K(+)-dependent reduction in steady-state inactivation. Mutation of two lysine residues in the outer vestibule of Kv2.1 (K356 and K382), to smaller, uncharged residues (glycine and valine, respectively), completely abolished K(+)-dependent potentiation that was not associated with inactivation. These mutations did not influence steady-state inactivation or the K(+)-dependent potentiation due to reduction in steady-state inactivation. These results demonstrate that K(+)-dependent potentiation can be completely accounted for by two independent mechanisms: one that involved the outer vestibule lysines and one that involved K(+)-dependent removal of channels from the inactivated state. Previous studies demonstrated that the outer vestibule of Kv2.1 can be in at least two conformations, depending on the occupancy of the selectivity filter by K(+) (Immke, D., M. Wood, L. Kiss, and S. J. Korn. 1999. J. Gen. Physiol. 113:819-836; Immke, D., and S. J. Korn. 2000. J. Gen. Physiol. 115:509-518). This change in conformation was functionally defined by a change in TEA sensitivity. Similar to the K(+)-dependent change in TEA sensitivity, the lysine-dependent potentiation depended primarily (>90%) on Lys-356 and was enhanced by lowering initial K(+) occupancy of the pore. Furthermore, the K(+)-dependent changes in current magnitude and TEA sensitivity were highly correlated. These results suggest that the previously described K(+)-dependent change in outer vestibule conformation underlies the lysine-sensitive, K(+)-dependent potentiation mechanism.

Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines

“…We used two well-characterized functional assays on two channels, the wild type Kv2.1 potassium channel and a mutant Kv2.1 channel that had two outer vestibule lysines neutralized (Kv2.1 K356G K382V). These two channels display different sensitivity to the extracellular channel blocker, tetraethylammonium (TEA) [6] and display different responsiveness to elevation of external [K + ] [7]. When over-expressed in HEK cells, these two channels are blocked by 3 mM external TEA by ~44% and ~81%, respectively ([6]; Fig.…”

“…Similar results were obtained for the K + -dependent change in current magnitude. When expressed in HEK cells, currents through Kv2.1 are potentiated by elevation of external [K + ] ([7]; Fig. 3D).…”

“…3D). In contrast, currents through the mutant channel are reduced by the same elevation of external [K + ] ([7]; Fig. 3D).…”

Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn2+

“…Additionally, a lysine residue (K356) which is positively charged at neutral pH, and which is homologous in position and charge to a protonated H463 of hKv1.5, is crucial in this K + ‐dependent conformational change (Immke et al 1999). The K356 residue is also involved in the enhancement of Kv2.1 currents by K + o (Wood & Korn, 2001).…”

Properties of ion channels in the protoplasts of the Mediterranean seagrass Posidonia oceanica

“…5c, where the two conductances, normalized to the maximum value measured in 450 m m K + , are compared. This paradoxic increase in channel conductance observed when decreasing the driving force has actually been observed in several other outward potassium channels both in animals and plants (Blatt 1991; Blatt & Gradmann 1997; Wood & Korn 2000; Ivashikina et al . 2001).…”