T-type Ca2+ channels and absence epilepsy

Burst firing of the thalamic neurons is driven by the low threshold Ca2+ spike generated by Ca2+ influx through T-type Ca2+ channels when these channels are activated by membrane hyperpolarization due to inhibitory inputs. The major inhibitory inputs to the thalamocortical (TC) neurons are from the GABAergic neurons in the thalamic reticular nucleus. Thalamic burst firings have long been implicated in the pathogenesis of absence epilepsy. The recent progress in genetic approaches has provided with an opportunity to examine this issue at the level of an organism. In this review I describe results primarily obtained from the analysis of the mice deficient for the alpha1G locus which is the predominant gene underlying the low threshold Ca2+ currents in the TC neurons. Current results so far demonstrate the essential role of the thalamocortical bursts in certain forms of absence seizures. Understanding of the pathophysiological mechanisms of absence epilepsy may help develop drugs to control the disease.     

Heteromeric K+ channels in plants

Voltage-gated potassium channels of plants are multimeric proteins built of four α-subunits. In the model plant Arabidopsis thaliana , nine genes coding for K⁺ channel α-subunits have been identified. When co-expressed in heterologous expression systems, most of them display the ability to form heteromeric K⁺ channels. Till now it was not clear whether plants use this potential of heteromerization to increase the functional diversity of potassium channels. Here, we designed an experimental approach employing different transgenic plant lines that allowed us to prove the existence of heteromeric K⁺ channels in plants. The chosen strategy might also be useful for investigating the activity and function of other multimeric channel proteins like, for instance, cyclic-nucleotide gated channels, tandem-pore K⁺ channels and glutamate receptor channels.     

Tethering chemistry and K+ channels

Voltage-gated K(+) channels are dynamic macromolecular machines that open and close in response to changes in membrane potential. These multisubunit membrane-embedded proteins are responsible for governing neuronal excitability, maintaining cardiac rhythmicity, and regulating epithelial electrolyte homeostasis. High resolution crystal structures have provided snapshots of K(+) channels caught in different states with incriminating molecular detail. Nonetheless, the connection between these static images and the specific trajectories of K(+) channel movements is still being resolved by biochemical experimentation. Electrophysiological recordings in the presence of chemical modifying reagents have been a staple in ion channel structure/function studies during both the pre- and post-crystal structure eras. Small molecule tethering agents (chemoselective electrophiles linked to ligands) have proven to be particularly useful tools for defining the architecture and motions of K(+) channels. This Minireview examines the synthesis and utilization of chemical tethering agents to probe and manipulate the assembly, structure, function, and molecular movements of voltage-gated K(+) channel protein complexes.     

Voltage-dependent K+ currents and underlying single K+ channels in pheochromocytoma cells

Properties of the whole-cell K+ currents and voltage-dependent activation and inactivation properties of single K+ channels in clonal pheochromocytoma (PC-12) cells were studied using the patch-clamp recording technique. Depolarizing pulses elicited slowly inactivating whole-cell K+ currents, which were blocked by external application of tetraethylammonium+, 4-aminopyridine, and quinidine. The amplitudes and time courses of these K+ currents were largely independent of the prepulse voltage. Although pharmacological agents and manipulation of the voltage-clamp pulse protocol failed to reveal any additional separable whole-cell currents in a majority of the cells examined, single-channel recordings showed that, in addition to the large Ca++-dependent K+ channels described previously in many other preparations, PC-12 cells had at least four distinct types of K+ channels activated by depolarization. These four types of K+ channels differed in the open-channel current-voltage relation, time course of activation and inactivation, and voltage dependence of activation and inactivation. These K+ channels were designated the Kw, Kz, Ky, and Kx channels. The typical chord conductances of these channels were 18, 12, 7, and 7 pS in the excised configuration using Na+-free saline solutions. These four types of K+ channels opened in the presence of low concentrations of internal Ca++ (1 nM). Their voltage-dependent gating properties can account for the properties of the whole-cell K+ currents in PC-12 cells.     

K+ channels and the microglial respiratory burst

Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K+ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K+ currents had properties of Kv1.3 and the Ca2+/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.     

钾通道家族新成员:双孔道钾通道的研究进展

双孔道钾通道（K2p）是钾通道超家族的新成员。它广泛分布于兴奋和非兴奋组织中,具有4次跨膜片段、两个孔道结构域的结构特征,目前主要分为：TWIK、多不饱和脂肪酸激活的钾通道、TASK和KCNK沉寂亚单位四类。K2p具有瞬间激活和不失活,以及对TEA、4－AP等经典钾通道阻断剂不敏感的电生理特性,参与调节背景钾电流或钾漏流。许多机械性和化学性刺激如细胞牵拉、pH值的变化、第二信使、花生四烯酸和吸入麻醉剂等均参与调控K2p通道。     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Na+ and K+ channels: history and structure

In this perspective, we discuss the physiological roles of Na and K channels, emphasizing the importance of the K channel for cellular homeostasis in animal cells and of Na and K channels for cellular signaling. We consider the structural basis of Na and K channel gating in light of recent structural and electrophysiological findings.     

Mechanism of rectification in inward-rectifier K+ channels

Rectification in inward-rectifier K+ channels is caused by the binding of intracellular cations to their inner pore. The extreme sharpness of this rectification reflects strong voltage dependence (apparent valence is approximately 5) of channel block by long polyamines. To understand the mechanism by which polyamines cause rectification, we examined IRK1 (Kir2.1) block by a series of bis-alkyl-amines (bis-amines) and mono-alkyl-amines (mono-amines) of varying length. The apparent affinity of channel block by both types of alkylamines increases with chain length. Mutation D172N in the second transmembrane segment reduces the channel's affinity significantly for long bis-amines, but only slightly for short ones (or for mono-amines of any length), whereas a double COOH-terminal mutation (E224G and E299S) moderately reduces the affinity for all bis-amines. The apparent valence of channel block increases from approximately 2 for short amines to saturate at approximately 5 for long bis-amines or at approximately 4 for long mono-amines. On the basis of these and other observations, we propose that to block the channel pore one amine group in all alkylamines tested binds near the same internal locus formed by the COOH terminus, while the other amine group of bis-amines, or the alkyl tail of mono-amines, "crawls" toward residue D172 and "pushes" up to 4 or 5 K+ ions outwardly across the narrow K+ selectivity filter. The strong voltage dependence of channel block therefore reflects the movement of charges carried across the transmembrane electrical field primarily by K+ ions, not by the amine molecule itself, as K+ ions and the amine blocker displace each other during block and unblock of the pore. This simple displacement model readily accounts for the classical observation that, at a given concentration of intracellular K+, rectification is apparently related to the difference between the membrane potential and the equilibrium potential for K+ ions rather than to the membrane potential itself.     

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.     

Molecular properties of voltage-gated K+ channels

Subfamilies of voltage-activated K⁺ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K⁺ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K⁺ selectivity have been identified. Susceptibility of certain K⁺ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K⁺ channels from mammalian brain. These are large (M_r 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()₄()₄, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for ₁, ₂ and ₃ subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of ₁ and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K⁺ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Apoptotic surface delivery of K+ channels

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.     

Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells

Electrical properties of the plasma membrane of guard cell protoplasts isolated from stomates of Vicia faba leaves were studied by application of the whole-cell configuration of the patch-clamp technique. The two types of K+ currents that have recently been identified in guard cells may allow efflux of K+ during stomatal closing, and uptake of K+ during stomatal opening (Schroeder et al., 1987). A detailed characterization of ion transport properties of the inward-rectifying (IK+,in) and the outward-rectifying (IK+,out) K+ conductance is presented here. The permeability ratios of IK+,in and IK+,out currents for K+ over monovalent alkali metal ions were determined. The resulting permeability sequences (PK+ greater than PRb+ greater than PNa+ greater than PLi+ much greater than PCs+) corresponded closely to the ion specificity of guard cell movements in V. faba. Neither K+ currents exhibited significant inactivation when K+ channels were activated for prolonged periods (greater than 10 min). The absence of inactivation may permit long durations of K+ fluxes, which occur during guard cell movements. Activation potentials of inward K+ currents were not shifted when external K+ concentrations were changed. This differs strongly from the behavior of inward-rectifying K+ channels in animal tissue. Blue light and fusicoccin induce hyperpolarization by stimulation of an electrogenic pump. From slow-whole-cell recordings it was concluded that electrogenic pumps require cytoplasmic substrates for full activation and that the magnitude of the pump current is sufficient to drive K+ uptake through IK+,in channels. First, direct evidence was gained for the hypothesis that IK+,in channels are a molecular pathway for K+ accumulation by the finding that IK+,in was blocked by Al3+ ions, which are known to inhibit stomatal opening but not closing. The results presented in this study strongly support a prominent role for IK+,in and IK+,out channels in K+ transport across the plasma membrane of guard cells.