Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a

KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.     

Kv Channel Gating Requires a Compatible S4-S5 Linker and Bottom Part of S6, Constrained by Non-interacting Residues

Voltage-dependent K⁺ channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6_T) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6_T. Exchanging both L45 and S6_T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6_T in hKv1.5 further shows that S6_T needs to be α-helical and forms a “crevice” in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6_T crevice, whereas other residues in S6_T and L45 that are not involved in the interaction maintain the correct structure of the coupling.     

Role of S4 positively charged residues in the regulation of Kv4.3 inactivation and recovery

The molecular and biophysical mechanisms by which voltage-sensitive K⁺ (Kv)4 channels inactivate and recover from inactivation are presently unresolved. There is a general consensus, however, that Shaker-like N- and P/C-type mechanisms are likely not involved. Kv4 channels also display prominent inactivation from preactivated closed states [closed-state inactivation (CSI)], a process that appears to be absent in Shaker channels. As in Shaker channels, voltage sensitivity in Kv4 channels is thought to be conferred by positively charged residues localized to the fourth transmembrane segment (S4) of the voltage-sensing domain. To investigate the role of S4 positive charge in Kv4.3 gating transitions, we analyzed the effects of charge elimination at each positively charged arginine (R) residue by mutation to the uncharged residue alanine (A). We first demonstrated that R290A, R293A, R296A, and R302A mutants each alter basic activation characteristics consistent with positive charge removal. We then found strong evidence that recovery from inactivation is coupled to deactivation, showed that the precise location of the arginine residues within S4 plays an important role in the degree of development of CSI and recovery from CSI, and demonstrated that the development of CSI can be sequentially uncoupled from activation by R296A, specifically. Taken together, these results extend our current understanding of Kv4.3 gating transitions. voltage-sensitive potassium channel; Shaker; closed-state inactivation     

Calculation of the Gating Charge for the Kv1.2 Voltage-Activated Potassium Channel

The atomic models of the Kv1.2 potassium channel in the active and resting state, originally presented elsewhere, are here refined using molecular dynamics simulations in an explicit membrane-solvent environment. With a minor adjustment of the orientation of the first arginine along the S4 segment, the total gating charge of the channel determined from >0.5 μs of molecular dynamics simulation is ∼12-12.7 e, in good accord with experimental estimates for the Shaker potassium channel, indicating that the final models offer a realistic depiction of voltage-gating. In the resting state of Kv1.2, the S4 segment in the voltage-sensing domain (VSD) spontaneously converts into a 3₁₀ helix over a stretch of 10 residues. The 3₁₀ helical conformation orients the gating arginines on S4 toward a water-filled crevice within the VSD and allows salt-bridge interactions with negatively charged residues along S2 and S3. Free energy calculations of the fractional transmembrane potential, acting upon key charged residues of the VSD, reveals that the applied field varies rapidly over a narrow region of 10-15 Å corresponding to the outer leaflet of the bilayer. The focused field allows the transfer of a large gating charge without translocation of S4 across the membrane.     

The Tetramerization Domain Potentiates Kv4 Channel Function by Suppressing Closed-State Inactivation

A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.     

The Tetramerization Domain Potentiates Kv4 Channel Function by Suppressing Closed-State Inactivation

A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.     

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.     

Gating Currents in the Hv1 Proton Channel

The Hv1 proton channel shares striking structural homology with fourth transmembrane helical segment-type voltage-sensor (VS) domains but manifests distinctive functional properties, including a proton-selective “aqueous” conductance and allosteric control of voltage-dependent gating by changes in the transmembrane pH gradient. The mechanisms responsible for Hv1’s functional properties remain poorly understood, in part because methods for measuring gating currents that directly report VS activation have not yet been described. Here, we describe an approach that allows robust and reproducible measurement of gating-associated charge movements in Hv1. Gating currents reveal that VS activation and proton-selective aqueous conductance opening are thermodynamically distinct steps in the Hv1 activation pathway and show that pH changes directly alter VS activation. The availability of an assay for gating currents in Hv1 may aid future efforts to elucidate the molecular mechanisms of gating cooperativity, pH-dependent modulation, and H⁺ selectivity in a model VS domain protein.     

Coupling of S4 Helix Translocation and S6 Gating Analyzed by Molecular-Dynamics Simulations of Mutated Kv Channels

The recently determined crystal structure of a chimeric Kv1.2-Kv2.1 Kv channel at 2.4 Å resolution motivated this molecular-dynamics simulation study of the chimeric channel and its mutants embedded in a DPPC membrane. For the channel protein, we used two types of C-terminus: E+ and Eo. E+ contains, and Eo lacks, the EGEE residue quartet located distal to the S6 helix. For both E+ and Eo, the following trend was observed: When S4 helices were restrained at the same position as in the x-ray structure (S4_high), the S6 gate remained open for 12 ns. The results were similar when the S4 helices were pulled downward 7 Å (S4_low). However, S4_middle (or S4_low) facilitated the S6 gate-narrowing for the following mutated channels (shown in order of increasing effect): 1), E395W; 2), E395W-F401A-F402A; and 3), E395W-F401A-F402A-V478W. The amino acid numbering system is that used for the Shaker channel. Even though all four subunits were set at S4_low, S6 gate-narrowing was often brought about by movements of only two opposing S6 helices toward the central axis of the pore, resulting in a twofold symmetry-like structure. A free-energy profile analysis over the ion conduction pathway shows that the two opposing S6 helices whose peptide backbones are ∼10.4 Å distant from each other lead to an energetic barrier of ∼25 kJ/mol. S6 movement was coupled with translocation of the S4-S5 linker toward the central axis of the same subunit, and the coupling was mediated by salt bridges formed between the inner (intracellular side) end of S4 and that of S6. Simulations in which S4 of only one subunit was pulled down to S4_low showed that a weak intersubunit coordination is present for S5 movement, whereas the coupling between the S4-S5 linker and S6 is largely an intrasubunit one. In general, whereas subunit-based behavior appears to be dominant and to permit heteromeric conformations of the pore domain, direct intersubunit coupling of S5 or S6 is weak. Therefore, the “concerted transition” of the pore domain that has been predicted based on electrophysiological analyses is likely to be mediated mainly by the dual effects of S4 and the S4-S5 linker; these segments of one subunit can interact with both S5 of the same subunit and that of the adjacent subunit.     

Independent and Cooperative Motions of the Kv1.2 Channel: Voltage Sensing and Gating

Voltage-gated potassium (Kv) channels, such as Kv1.2, are involved in the generation and propagation of action potentials. The Kv channel is a homotetramer, and each monomer is composed of a voltage-sensing domain (VSD) and a pore domain (PD). We analyzed the fluctuations of a model structure of Kv1.2 using elastic network models. The analysis suggested a network of coupled fluctuations of eight rigid structural units and seven hinges that may control the transition between the active and inactive states of the channel. For the most part, the network is composed of amino acids that are known to affect channel activity. The results suggested allosteric interactions and cooperativity between the subunits in the coupling between the motion of the VSD and the selectivity filter of the PD, in accordance with recent empirical data. There are no direct contacts between the VSDs of the four subunits, and the contacts between these and the PDs are loose, suggesting that the VSDs are capable of functioning independently. Indeed, they manifest many inherent fluctuations that are decoupled from the rest of the structure. In general, the analysis suggests that the two domains contribute to the channel function both individually and cooperatively.     

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg^3′), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg^3′ in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu⁴⁵ in non-α-subunits. In all subunits, mutations of Arg^3′ had similar effects as in the α-subunit. In the ϵ-subunit, mutations of the flanking residues and Glu⁴⁵ had only small effects, and there was no energy coupling between ϵGlu⁴⁵ and ϵArg^3′. The non-α-subunit Arg^3′ residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu⁴⁵, but only in the α-subunits.     

Contribution of electrostatic and structural properties of Kv4.3 S4 arginine residues to the regulation of channel gating

Previous work has demonstrated that replacing individual arginine (R) residues in the S4 domain of Kv4.3 with alanine (A) not only altered activation and deactivation processes, but also those of closed-state inactivation (CSI) and recovery. R → A mutants eliminated individual positive charge while substantially reducing side chain volume and hydrophilic character. Their novel effects on gating may thus have been the result of electrostatic and/or structural perturbations. To address this issue, and to gain further insights into the roles that S4 plays in the regulation of Kv4.3 gating transitions, we comparatively analyzed arginine to glutamine (R → Q) mutations at positions 290, 293, and 296. This maneuver maintained positive charge elimination of the R → A mutants, while partially restoring native side chain volume and hydrophilic properties. R → A and R → Q mutant pairs produced similar effects on the forward gating process of activation. In contrast, significant differences between the two substitutions were discovered on deactivation, CSI, and recovery, with the R → Q mutants partially restoring wild type characteristics. Our results argue that modification of individual S4 residue properties may result in altered localized interactions within unique microenvironments encountered during forward and reverse gating transitions. As such, predominant effects appear on the reverse gating transitions of deactivation and recovery. These results are consistent with the proposal that arginine residues in S4 are involved in regulating Kv4.3 CSI and recovery.     

Gating Transitions in the Palm Domain of ASIC1a

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.     

Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K⁺ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons.     

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.     

S1 constrains S4 in the voltage sensor domain of Kv7.1 K+ channels

Voltage-gated K(+) channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (approximately 190 degrees) and outward translation of S4 (approximately 12 A) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1-S4 interaction to an inter-VSD S1-S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating.     

Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Interdomain interactions between intracellular N and C termini have been described for various K⁺ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the “docking loop” at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.Interdomain interactions between intracellularly located N and C termini have been described for various K⁺ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca²⁺-activated (hSK3) (3), and voltage-gated Kv2.1 (4) and Kv4.1 (5) channels. In the case of Kv2.1, two modes of interaction have been proposed: an association of the distal part of Kv2.1 C terminus (termed CTA domain; amino acids (aa) 741–853)⁴ with aa 67 and 75 of the Kv2.1 N terminus (4); or an association between the proximal part of the Kv2.1 C terminus (aa 444–477) and the predicted loop structure (aa 55–71) in the N-terminal T1 domain (6). In addition, involvement of the S4-S5 linker in this interaction has been suggested (7). Although these studies propose two different C-terminal sites, they indicate a specific loop in the N terminus of Kv2.1 (6, 8), which could be functionally related to the Shaker and Shal docking loops in the lateral part of their T1 domains (9, 10). These latter loops are responsible for the subfamily-specific association with β-subunits (Kvβ and KChIP, respectively). Further, the interaction between the N and C cytoplasmic termini (N/C interaction) of Kv2.1 has been shown to be dynamic and voltage-dependent and to involve structural rearrangements between these domains, which could affect both activation and inactivation gating of the channel (4, 6, 7). These rearrangements can be clearly detected with fluorescence resonance energy transfer (FRET) (11). A similar N/C interaction has been shown to affect gating of the closely related Kv4.1 channel (5, 12).It is conceivable that the specific packaging of Kv2.1 cytoplasmic termini (a relatively long C terminus (>400 aa) wrapping the N terminus (<190 aa) from the outside (4)) not only supports multiple interactions between the termini but also reflects the fact that most of the interactions of the channel with intracellular and membrane-bound regulatory factors occur at the C terminus, including channel phosphorylation (13–15), clustering through a unique proxinal restriction and clustering signal (16), and protein-protein interactions with both the plasma membrane SNAREs, syntaxin 1A and SNAP-25 (17–19), and the MiRP2 (KCNE3) peptide (20). For the Kv2.1 N terminus, on the other hand, there are only two examples of protein-protein interactions: a transient association with KChAP (21), which does not affect channel function; and an interaction with the vesicular SNARE partner VAMP2 (synaptobrevin 2), which is also present at a high concentration in the neuronal plasma membrane and enhances channel inactivation (8). Specifically, VAMP2 has been shown to associate with the extension of a docking loop in the lateral part of the T1 domain (8) near the site of interaction with the C terminus (4, 6). Thus, it is reasonable to hypothesize that interaction with VAMP2 will affect the N/C interaction, similar to proton-mediated Kir2.3 (1) and Kir1.1 (22) N/C interactions or the ATP-dependent Kir6.2 (2) N/C interaction. To date, no protein molecule that directly affects N/C interactions in a K⁺ channel has been demonstrated. Because VAMP2 was the first protein documented to affect Kv2.1 channel gating by binding to a specific N-terminal site, which is probably masked by the C terminus, we have put forward the idea that its interaction with the Kv2.1 N terminus requires conformational changes in the C terminus that will enable its access to the N terminus.Here we endeavored to gain a mechanistic and structural understanding of the Kv2.1-VAMP2 interaction. Based on our evidence, we propose that VAMP2 modulates Kv2.1 gating by interfering with the Kv2.1 cytoplasmic N/C interaction.     

Multi-Walled Carbon Nanotubes Impair Kv4.2/4.3 Channel Activities,Delay Membrane Repolarization and Induce Bradyarrhythmias in the Rat

Purpose

The potential hazardous effects of multi-walled carbon nanotubes (MWCNTs) on cardiac electrophysiology are seldom evaluated. This study aimed to investigate the impacts of MWCNTs on the Kv4/I _to channel, action potential and heart rhythm and the underlying mechanisms.

Methods

HEK293 cells were engineered to express Kv4.2 or Kv4.3 with or without KChIP2 expression. A series of approaches were introduced to analyze the effects of MWCNTs on Kv4/I _to channel kinetics, current densities, expression and trafficking. Transmission electron microscopy was performed to observe the internalization of MWCNTs in HEK293 cells and rat cardiomyocytes. Current clamp was employed to record the action potentials of isolated rat cardiomyocytes. Surface ECG and epicardial monophasic action potentials were recorded to monitor heart rhythm in rats in vivo. Vagal nerve discharge monitoring and H&E staining were also performed.

Results

Induction of MWCNTs into the cytosole through pipette solution soon accelerated the decay of I _Kv4 in HEK293 cells expressing Kv4.2/4.3 and KChIP2, and promoted the recovery from inactivation when Kv4.2 or Kv4.3 was expressed alone. Longer exposure (6 h) to MWCNTs decreased the I _Kv4.2 density, Kv4.2/Kv4.3 (but not KChIP2) expression and trafficking towards the plasma membrane in HEK293 cells. In acutely isolated rat ventricular myocytes, pipette MWCNTs also quickly accelerated the decay of I _Kv4 and prolonged the action potential duration (APD). Intravenous infusion of MWCNTs (2 mg/rat) induced atrioventricular (AV) block and even cardiac asystole. No tachyarrhythmia was observed after MWCNTs administration. MWCNTs did not cause coronary clot but induced myocardial inflammation and increased vagus discharge.

Conclusions

MWCNTs suppress Kv4/I _to channel activities likely at the intracellular side of plasma membrane, delay membrane repolarization and induce bradyarrhythmia. The delayed repolarization, increased vagus output and focal myocardial inflammation may partially underlie the occurrence of bradyarrhythmias induced by MWCNTs. The study warns that MWCNTs are hazardous to cardiac electrophysiology.