Linda M. Boland

Inhibitory effects of polyunsaturated fatty acids on Kv4/KChIP potassium channels.

Kv4/K channel interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K(+) channels in neurons and cardiac muscle. Modulation of Kv4/KChIP channels by polyunsaturated fatty acids (PUFAs) is important in the regulation of cellular excitability and the induction of activity-dependent synaptic plasticity. Using the Xenopus laevis oocyte expression system, we studied the inhibition by PUFAs of the peak outward K(+) current and the accompanying increase in the rate of current inactivation of rKv4.2/rKChIP1b. Inhibitory effects do not depend on KChIP coexpression since Kv4.2 channels lacking an NH(2)-terminal KChIP association region were substantially inhibited by PUFAs and showed strong kinetic modulation. PUFAs accelerated both the fast and slow time constants that describe the kinetics of Kv4/KChIP inactivation. The time course of entry into closed inactivated states was facilitated by PUFAs, but steady-state inactivation and recovery from inactivation were unaltered. PUFA inhibition of Kv4/KChIP current was not use dependent. The concentration-response relationship for arachidonic acid (AA) inhibition of Kv4/KChIP channels mimicked that for activation of TRAAK channels. Internal serum albumin largely prevents the inhibitory effects of externally applied AA, and the membrane-impermeant AA-CoA is inactive when applied externally. Overall, our data suggest that PUFAs inhibit Kv4/KChIP channels by facilitating inactivation from open and closed gating states and that access of the fatty acid to the internal leaflet of the membrane is important. These results improve our understanding of the mechanisms for the inhibitory effects of PUFAs on Kv4/KChIP channel function.

Expression of a poriferan potassium channel: insights into the evolution of ion channels in metazoans

SUMMARY Ion channels establish and regulate membrane potentials in excitable and non-excitable cells. How functional diversification of ion channels contributed to the evolution of nervous systems may be understood by studying organisms at key positions in the evolution of animal multicellularity. We have carried out the first analysis of ion channels cloned from a marine sponge, Amphimedon queenslandica. Phylogenetic comparison of sequences encoding for poriferan inward-rectifier K+ (Kir) channels suggests that Kir channels from sponges, cnidarians and triploblastic metazoans each arose from a single channel and that duplications arose independently in the different groups. In Xenopus oocytes, AmqKirA and AmqKirB produced K+ currents with strong inward rectification, as seen in the mammalian Kir2 channels, which are found in excitable cells. The pore properties of AmqKir channels demonstrated strong K+ selectivity and block by Cs+ and Ba2+. We present an original analysis of sponge ion channel physiology and an examination of the phylogenetic relationships of this channel with other cloned Kir channels.

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca2+-activated Cl− channels is controlled by a complex interplay among [Ca2+]i, membrane potential and permeant anions. Besides Ca2+, Ba2+ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca2+ is most effective, followed by Sr2+ and Ni2+, which have similar affinity, while Mg2+ is ineffective. Zn2+ does not activate TMEM16A but inhibits the Ca2+-activated chloride currents. Maximally effective concentrations of Sr2+ and Ni2+ occluded activation of the TMEM16A current by Ca2+, which suggests that Ca2+, Sr2+ and Ni2+ all regulate the channel by the same mechanism.

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels.

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K+ currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K+ currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

A Xenopus oocyte model system to study action potentials

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and “temporally shaping” the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression.