A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 ± 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels.

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.

Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

Ca2+-activated K+(BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K+ currents that contribute to action potential repolarisation. In different neuronal cell types these K+ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K+ currents. Recent findings show that inactivation of a Ca2+-dependent K+ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca2+ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or alpha-dendrotoxin. We conclude that while inactivation of BK-type Ca2+-activated K+ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.

Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells

K+ Channels and Membrane Potential in Endothelial Cells. The endothelium plays a vital role in the control of vascular functions, including modulation of tone; permeability and barrier properties; platelet adhesion and aggregation; and secretion of paracrine factors. Critical signaling events in many of these functions involve an increase in intracellular free Ca2+ concentration ([Ca2+](i)). This rise in [Ca2+](i) occurs via an interplay between several mechanisms, including release from intracellular stores, entry from the extracellular space through store depletion and second messenger-mediated processes, and the establishment of a favorable electrochemical gradient. The focus of this review centers on the role of potassium channels and membrane potential in the creation of a favorable electrochemical gradient for Ca2+ entry. In addition, evidence is examined for the existence of various classes of potassium channels and the possible influence of regional variation in expression and experimental conditions.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na-v,.) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na-v channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na-v currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that Occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP. and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na-v. channel gating, observed clinically in response to ciguatera poisoning. (c) 2004 Elsevier Inc. All rights reserved.

SK channels regulate excitatory synaptic transmission and plasticity in the lateral amygdala

At glutamatergic synapses, calcium influx through NMDA receptors (NMDARs) is required for long-term potentiation (LTP); this is a proposed cellular mechanism underlying memory and learning. Here we show that in lateral amygdala pyramidal neurons, SK channels are also activated by calcium influx through synaptically activated NMDARs, resulting in depression of the synaptic potential. Thus, blockade of SK channels by apamin potentiates fast glutamatergic synaptic potentials. This potentiation is blocked by the NMDAR antagonist AP5 (D(-)-2-amino-5-phosphono-valeric acid) or by buffering cytosolic calcium with BAPTA. Blockade of SK channels greatly enhances LTP of cortical inputs to lateral amygdala pyramidal neurons. These results show that NMDARs and SK channels are colocalized at glutamatergic synapses in the lateral amygdala. Calcium influx through NMDARs activates SK channels and shunts the resultant excitatory postsynaptic potential. These results demonstrate a new role for SK channels as postsynaptic regulators of synaptic efficacy.

Epistatic effects of potassium channel variation on cardiac repolarization and atrial fibrillation risk

Objectives The aim of this study was to evaluate the role of cardiac K+ channel gene variants in families with atrial fibrillation (AF). Background The K+ channels play a major role in atrial repolarization but single mutations in cardiac K+ channel genes are infrequently present in AF families. The collective effect of background K+ channel variants of varying prevalence and effect size on the atrial substrate for AF is largely unexplored. Methods Genes encoding the major cardiac K+ channels were resequenced in 80 AF probands. Nonsynonymous coding sequence variants identified in AF probands were evaluated in 240 control subjects. Novel variants were characterized using patch-clamp techniques and in silico modeling was performed using the Courtemanche atrial cell model. Results Nineteen nonsynonymous variants in 9 genes were found, including 11 rare variants. Rare variants were more frequent in AF probands (18.8% vs. 4.2%, p < 0.001), and the mean number of variants was greater (0.21 vs. 0.04, p < 0.001). The majority of K+ channel variants individually had modest functional effects. Modeling simulations to evaluate combinations of K+ channel variants of varying population frequency indicated that simultaneous small perturbations of multiple current densities had nonlinear interactions and could result in substantial (>30 ms) shortening or lengthening of action potential duration as well as increased dispersion of repolarization. Conclusions Families with AF show an excess of rare functional K+ channel gene variants of varying phenotypic effect size that may contribute to an atrial arrhythmogenic substrate. Atrial cell modeling is a useful tool to assess epistatic interactions between multiple variants.

Involvement of Ca2+-dependent hyperpolarization in sleep duration in mammals

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca2+-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca2+-dependent K+ channels (Kcnn2 and Kcnn3), voltage-gated Ca2+ channels (Cacna1g and Cacna1h), or Ca2+/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca2+ ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca2+-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.