Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.     

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.Presented at the Biophysical Society Meeting on Ion channels—from structure to disease held in May 2003, Rennes, France     

The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism

Single calcium dependent potassium channels from cultured rat myoballs have been studied with the patch clamp technique, and current records subjected to statistical analysis. From the dependence of the mean open state probability on the internal calcium concentration, two calcium ions are required to open the channel. The open state and closed state lifetime distributions reveal that the usual activation model is not applicable to these channels. They are consistent with a two step gating mechanism that involves both activation by calcium and blockade by a calcium-sensitive gate.     

Do Lipids Show State-dependent Affinity to the Voltage-gated Potassium Channel KvAP?

As all integral membrane proteins, voltage-gated ion channels are embedded in a lipid matrix that regulates their channel behavior either by physicochemical properties or by direct binding. Because manipulation of the lipid composition in cells is difficult, we investigated the influence of different lipids on purified KvAP channels reconstituted in planar lipid bilayers of known composition. Lipids developed two distinct and independent effects on the KvAP channels; lipids interacting with the pore lowered the energy barriers for the final transitions, whereas voltage sensor-bound lipids shifted the midpoint of activation dependent on their electrostatic charge. Above all, the midpoint of activation was determined only by those lipids the channels came in contact with first after purification and can seemingly only be exchanged if the channel resides in the open state. The high affinity of the bound lipids to the binding site has implications not only on our understanding of the gating mechanism but also on the general experimental design of any lipid dependence study.     

Properties of Shaker-type Potassium Channels in Higher Plants

Potassium (K⁺), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na⁺/K⁺ exchanger, which widely exists in animal cells, K⁺ channels and some type of K⁺ transporters function as K⁺ uptake systems in plants. Plant voltage-dependent K⁺ channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K⁺ channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K⁺ channels have been identified and play a crucial role in K⁺ homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K⁺ channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K⁺ channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K⁺ channels in plants in comparison to those of Shaker channels in animals and bacteria.     

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.     

Ionic channels,ion transport and plant cell membranes: Potential applications of the patch-clamp technique

Conclusion Exciting innovations in the methodologies available for the study of ionic channels (notably in animal cells) have allowed hitherto impossible advances in the comprehension of both structure and function. In using channels like the Na channel and the AChR as examples of these strategies, we have tried to give a concise but up to date account of the current possibilities (in particular, the patch-clamp) for research in membrane physiology. That few of these techniques have been applied to plant cell membranes simply indicates the scope for advancement in the understanding of some problems fundamental to plant physiology. The mechanisms of transport involved in processes known to be important for the life of plant cells (e.g., regulation of cytoplasmic and vacuolar potential differences and pH, maintenance of vacuolar turgor pressure, accumulation of metabolites and their counterions, response to environmental stimuli) are relatively speaking, poorly characterized. In that ion fluxes through plasmalemma and tonoplast membranes are at least in part likely to be via ionic channels for all of these processes, an important step forward would be the application of patch-clamp techniques for the direct demonstration of a channel mechanism and the subsequent elucidation of their role.     

Potassium channels

The atomic structures of K+ channels have added a new dimension to our understanding of K+ channel function. I will briefly review how structures have influenced our views on ion conduction, gating of the pore, and voltage sensing.     

Intracellular regions of the Eag potassium channel play a critical role in generation of voltage-dependent currents

Folding, assembly, and trafficking of ion channels are tightly controlled processes and are important for biological functions relevant to health and disease. Here, we report that functional expression of the Eag channel is temperature-sensitive by a mechanism that is independent of trafficking or surface targeting of the channel protein. Eag channels in cells grown at 37 °C exhibit voltage-evoked gating charge movements but fail to conduct K(+) ions. By mutagenesis and chimeric channel studies, we show that the N- and C-terminal regions are involved in controlling a step after movement of the voltage sensor, as well as in regulating biophysical properties of the Eag channel. Synthesis and assembly of Eag at high temperature disrupt the ability of these domains to carry out their function. These results suggest an important role of the intracellular regions in the generation of Eag currents.     

Scanning the Topography of Polyamine Blocker Binding in an Inwardly Rectifying Potassium Channel

Steeply voltage-dependent inward rectification of Kir (inwardly rectifying potassium) channels arises from blockade by cytoplasmic polyamines. These polycationic blockers traverse a long (>70 Å) pore, displacing multiple permeant ions, en route to a high affinity binding site that remains loosely defined. We have scanned the effects of cysteine modification at multiple pore-lining positions on the blocking properties of a library of polyamine analogs, demonstrating that the effects of cysteine modification are position- and blocker-dependent. Specifically, introduction of positively charged adducts results in two distinct phenotypes: either disruption of blocker binding or generation of a barrier to blocker migration, in a consistent pattern that depends on both the length of the polyamine blocker and the position of the modified cysteine. These findings reveal important details about the chemical basis and specific location of high affinity polyamine binding.     

RNA Editing in the Central Cavity as a Mechanism to Regulate Surface Expression of the Voltage-gated Potassium Channel Kv1.1

Voltage-gated potassium (Kv) 1.1 channels undergo a specific enzymatic RNA deamination, generating a channel with a single amino acid exchange located in the inner pore cavity (Kv1.1^I400V). We studied I400V-edited Kv1.1 channels in more detail and found that Kv1.1^I400V gave rise to much smaller whole-cell currents than Kv1.1. To elucidate the mechanism behind this current reduction, we conducted electrophysiological recordings on single-channel level and did not find any differences. Next we examined channel surface expression in Xenopus oocytes and HeLa cells using a chemiluminescence assay and found the edited channels to be less readily expressed at the surface membrane. This reduction in surface expression was verified by fluorescence imaging experiments. Western blot analysis for comparison of protein abundances and glycosylation patterns did not show any difference between Kv1.1 and Kv1.1^I400V, further indicating that changed trafficking of Kv1.1^I400V is causing the current reduction. Block of endocytosis by dynasore or AP180C did not abolish the differences in current amplitudes between Kv1.1 and Kv1.1^I400V, suggesting that backward trafficking is not affected. Therefore, our data suggest that I400V RNA editing of Kv1.1 leads to a reduced current size by a decreased forward trafficking of the channel to the surface membrane. This effect is specific for Kv1.1 because coexpression of Kv1.4 channel subunits with Kv1.1^I400V abolishes these trafficking effects. Taken together, we identified RNA editing as a novel mechanism to regulate homomeric Kv1.1 channel trafficking. Fine-tuning of Kv1.1 surface expression by RNA editing might contribute to the complexity of neuronal Kv channel regulation.     

Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels

Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the “slide helix” forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.     

植物钾营养高效与膜运系统的关系

HKT1和HAK1等转运子介导钾离子的高亲和吸收以及K^ /Na^ 共运转,从而可能增强Na^ 替代K^ 能力,KAT1和KST1等离子通道介导钾离子的累积和转运,从而调节气孔细胞的渗透压,控制气孔运动,阐述了植物生物膜上离子转运机制和钾营养高效机理的某种可能的关系,这些转运子和通道的高效表达可能与植物钾营养高效有很大的相关性。     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

Plasmodium falciparum-induced channels

To survive within a red blood cell, the malaria parasite alters dramatically the permeability of the host's plasma membrane (allowing the uptake of essential nutrients and the removal of potentially hazardous metabolites). The pathway(s) responsible for the increased permeability have been proposed as putative chemotherapeutic targets and/or selective routes for antimalarial agents that target the internal parasite. This review covers our current understanding of this parasite-induced phenomenon in Plasmodium falciparum-infected human red blood cells. In particular, recent electrophysiological studies, using the patch-clamp technique, are reviewed.     

Modulation of outward K+ conductance is a post-activational event in rat T lymphocytes responsible for the adoptive transfer of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is an accepted animal model for the human demyelinating disease multiple sclerosis. The continuously propagated line of Lewis rat T helper lymphocytes (GP1 T cells), specific for the encephalitogenic 68–86 sequence of guinea pig myelin basic protein (GPMBP), mediates the adoptive transfer of EAE into normal syngeneic Lewis rats. Because mitogenic activation of T cells can increase K⁺ conductance, this study investigated changes in the outwardly rectifying K⁺ conductance in GP1 T cells following activation with the encephalitogen, GPMBP. Using the gigohm-seal whole-cell variation of the patch clamp technique, GP1 T cells were studied during a 3-day culture with GPMBP and throughout the subsequent 10 days, as cells progressed through both GPMBP-induced activation (EAE transfer activity) and proliferation responses, finally reverting to the resting state. Resting GP1 T cells exhibited peak K⁺ conductances around 2 nS, while GPMBP-induced activation resulted in 5- to 10-fold increases in peak K⁺ conductance, which temporally coincided with the optimal period of EAE transfer activity. During and immediately after the optimal period for EAE transfer, 20-mV depolarizing shifts in the voltage dependence of both activation and inactivation developed, abruptly reversing to resting values as cells reverted to the resting state. Accompanying the depolarizing shifts were a slowing of the K⁺ current activation kinetics and an acceleration of the deactivation kinetics. These results indicate that the K⁺ conductance in GP1 rat T helper cells is modulated over the full time course of GPMBP-induced cellular responses and that K⁺ channels should be optimally available during the period of adoptive EAE transfer, preceding disease manifestation.     

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.