BACKGROUND: \'Hongyang\' kiwifruit (Actinidia chinensis cv \'Hongyang\') is a high-quality variety of A. chinensis with the advantages of high yield, early ripening, and high stress tolerance. Studies have confirmed that the Shaker K+ genes family is involved in plant uptake and distribution of potassium (K+).
RESULTS: Twenty-eight Shaker genes were identified and analyzed from the \'Hongyang\' kiwifruit (A. chinensis cv \'Hongyang\') genome. Subcellular localization results showed that more than one-third of the AcShaker genes were on the cell membrane. Phylogenetic analysis indicated that the AcShaker genes were divided into six subfamilies (I-VI). Conservative model, gene structure, and structural domain analyses showed that AcShaker genes of the same subfamily have similar sequence features and structure. The promoter cis-elements of the AcShaker genes were classified into hormone-associated cis-elements and environmentally stress-associated cis-elements. The results of chromosomal localization and duplicated gene analysis demonstrated that AcShaker genes were distributed on 18 chromosomes, and segmental duplication was the prime mode of gene duplication in the AcShaker family. GO enrichment analysis manifested that the ion-conducting pathway of the AcShaker family plays a crucial role in regulating plant growth and development and adversity stress. Compared with the transcriptome data of the control group, all AcShaker genes were expressed under low-K+stress, and the expression differences of three genes (AcShaker15, AcShaker17, and AcShaker22) were highly significant. The qRT-PCR results showed a high correlation with the transcriptome data, which indicated that these three differentially expressed genes could regulate low-K+ stress and reduce K+ damage in kiwifruit plants, thus improving the resistance to low-K+ stress. Comparison between the salt stress and control transcriptomic data revealed that the expression of AcShaker11 and AcShaker18 genes was significantly different and lower under salt stress, indicating that both genes could be involved in salt stress resistance in kiwifruit.
CONCLUSIONS: The results showed that 28 AcShaker genes were identified and all expressed under low K+ stress, among which AcShaker22 was differentially and significantly upregulated. The AcShaker22 gene can be used as a candidate gene to cultivate new varieties of kiwifruit resistant to low K+ and provide a reference for exploring more properties and functions of the AcShaker genes.

Plants often face high salinity as a significant environmental challenge with roots being the first responders to this stress. Maintaining K+/Na+ ratio within plant cells is crucial for survival, as the intracellular K+ level decreases and the intracellular Na+ level increases under saline conditions. However, knowledge about the molecular regulatory mechanisms of K+ loss in response to salt stress through outward-rectifying K+ channels in plants is largely unknown. In this study, we found that the Arabidopsis double mutant gorkskor, in which the GORK and SKOR genes are disrupted, showed an improved primary root growth under salt stress compared to wild-type (WT) and the gork and skor single-mutant plants. No significant differences in the sensitivity to mannitol stress between the WT and gorkskor mutant were observed. Accumulation of ROS induced by salt stress was reduced in the gorkskor roots. The gorkskor mutant seedlings had significantly higher K+ content, lower Na+ content, and a greater resultant K+/Na+ ratio than the WT under salt stress. Moreover, salt-stress-induced elevation of cytosolic free Ca2+ concentration was reduced in the gorkskor roots. Taken together, these results suggest that Arabidopsis Shaker-type outward-rectifying K+ channels GORK and SKOR may redundantly function in regulation of primary root growth under salt stress and are involved in not only the late-stage response (e.g. K+ leakage) but also the early response including ROS production and [Ca2+]cyt elevation.

The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.

The voltage-dependent potassium channels (Kv channels) show several different types of inactivation. N-type inactivation is a fast inactivating mechanism, which is essentially an open pore blockade by the amino-terminal structure of the channel itself or the auxiliary subunit. There are several functionally discriminatable slow inactivation (C-type, P-type, U-type), the mechanism of which is supposed to include rearrangement of the pore region. In some Kv1 channels, the actual inactivation is brought about by coupling of N-type and C-type inactivation (N-C coupling). In the present study, we focused on the N-C coupling of the Aplysia Kv1 channel (AKv1). AKv1 shows a robust N-type inactivation, but its recovery is almost thoroughly from C-type inactivated state owing to the efficient N-C coupling. In the I8Q mutant of AKv1, we found that the inactivation as well as its recovery showed two kinetic components apparently correspond to N-type and C-type inactivation. Also, the cumulative inactivation which depends on N-type mechanism in AKv1 was hindered in I8Q, suggesting that N-type inactivation of I8Q is less stable. We also found that Zn 2 + specifically accelerates C-type inactivation of AKv1 and that H382 in the pore turret is involved in the Zn 2 + binding. Because the region around Ile 8 (I8) in AKv1 has been suggested to be involved in the pre-block binding of the amino-terminal structure, our results strengthen a hypothesis that the stability of the pre-block state is important for stable N-type inactivation as well as the N-C coupling in the Kv1 channel inactivation.

Persistent activity in excitatory pyramidal cells (PYRs) is a putative mechanism for maintaining memory traces during working memory. We have recently demonstrated persistent interruption of firing in fast-spiking parvalbumin-expressing interneurons (PV-INs), a phenomenon that could serve as a substrate for persistent activity in PYRs through disinhibition lasting hundreds of milliseconds. Here, we find that hippocampal CA1 PV-INs exhibit type 2 excitability, like striatal and neocortical PV-INs. Modeling and mathematical analysis showed that the slowly inactivating potassium current KV1 contributes to type 2 excitability, enables the multiple firing regimes observed experimentally in PV-INs, and provides a mechanism for robust persistent interruption of firing. Using a fast/slow separation of times scales approach with the KV1 inactivation variable as a bifurcation parameter shows that the initial inhibitory stimulus stops repetitive firing by moving the membrane potential trajectory onto a coexisting stable fixed point corresponding to a nonspiking quiescent state. As KV1 inactivation decays, the trajectory follows the branch of stable fixed points until it crosses a subcritical Hopf bifurcation (HB) and then spirals out into repetitive firing. In a model describing entorhinal cortical PV-INs without KV1, interruption of firing could be achieved by taking advantage of the bistability inherent in type 2 excitability based on a subcritical HB, but the interruption was not robust to noise. Persistent interruption of firing is therefore broadly applicable to PV-INs in different brain regions but is only made robust to noise in the presence of a slow variable, KV1 inactivation.

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel\'s voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Here we explore the evolutionary origins of fast N-type ball-and-chain inactivation in Shaker (Kv1) K+ channels by functionally characterizing Shaker channels from the ctenophore (comb jelly) Mnemiopsis leidyi. Ctenophores are the sister lineage to other animals and Mnemiopsis has >40 Shaker-like K+ channels, but they have not been functionally characterized. We identified three Mnemiopsis channels (MlShak3-5) with N-type inactivation ball-like sequences at their N termini and functionally expressed them in Xenopus oocytes. Two of the channels, MlShak4 and MlShak5, showed rapid inactivation similar to cnidarian and bilaterian Shakers with rapid N-type inactivation, whereas MlShak3 inactivated ∼100-fold more slowly. Fast inactivation in MlShak4 and MlShak5 required the putative N-terminal inactivation ball sequences. Furthermore, the rate of fast inactivation in these channels depended on the number of inactivation balls/channel, but the rate of recovery from inactivation did not. These findings closely match the mechanism of N-type inactivation first described for Drosophila Shaker in which 1) inactivation balls on the N termini of each subunit can independently block the pore, and 2) only one inactivation ball occupies the pore binding site at a time. These findings suggest classical N-type activation evolved in Shaker channels at the very base of the animal phylogeny in a common ancestor of ctenophores, cnidarians, and bilaterians and that fast-inactivating Shakers are therefore a fundamental type of animal K+ channel. Interestingly, we find evidence from functional co-expression experiments and molecular dynamics that MlShak4 and MlShak5 do not co-assemble, suggesting that Mnemiopsis has at least two functionally independent N-type Shaker channels.

Shaker potassium channels have been an essential model for studying inactivation of ion channels and shaped our earliest understanding of N-type vs. C-type mechanisms. In early work describing C-type inactivation, López-Barneo and colleagues systematically characterized numerous mutations of Shaker residue T449, demonstrating that this position was a key determinant of C-type inactivation rate. In most of the closely related mammalian Kv1 channels, however, a persistent enigma has been that residue identity at this position has relatively modest effects on the rate of inactivation in response to long depolarizations. In this study, we report alternative ways to measure or elicit conformational changes in the outer pore associated with C-type inactivation. Using a strategically substituted cysteine in the outer pore, we demonstrate that mutation of Kv1.2 V381 (equivalent to Shaker T449) or W366 (Shaker W434) markedly increases susceptibility to modification by extracellularly applied MTSET. Moreover, due to the cooperative nature of C-type inactivation, Kv1.2 assembly in heteromeric channels markedly inhibits MTSET modification of this substituted cysteine in neighboring subunits. The identity of Kv1.2 residue V381 also markedly influences function in conditions that bias channels toward C-type inactivation, namely when Na+ is substituted for K+ as the permeant ion or when channels are blocked by an N-type inactivation particle (such as Kvβ1.2). Overall, our findings illustrate that in mammalian Kv1 channels, the identity of the T449-equivalent residue can strongly influence function in certain experimental conditions, even while having modest effects on apparent inactivation during long depolarizations. These findings contribute to reconciling differences in experimental outcomes in many Kv1 channels vs. Shaker.

Ion channels are transmembrane structures that allow the passage of ions across cell membranes such as the plasma membrane or the membranes of various organelles like the nucleus, endoplasmic reticulum, Golgi apparatus or mitochondria. Aberrant expression of various ion channels has been demonstrated in several tumor cells, leading to the promotion of key functions in tumor development, such as cell proliferation, resistance to apoptosis, angiogenesis, invasion and metastasis. The link between ion channels and these key biological functions that promote tumor development has led to the classification of cancers as oncochannelopathies. Among all ion channels, the most varied and numerous, forming the largest family, are the potassium channels, with over 70 genes encoding them in humans. In this context, this review will provide a non-exhaustive overview of the role of plasma membrane potassium channels in cancer, describing 1) the nomenclature and structure of potassium channels, 2) the role of these channels in the control of biological functions that promotes tumor development such as proliferation, migration and cell death, and 3) the role of two particular classes of potassium channels, the SKCa- and Kv1- type potassium channels in cancer progression.