SGTx1, a Kv channel gating-modifier toxin, binds to the interfacial region of lipid bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.     

Lipid bilayer pressure profiles and mechanosensitive channel gating

The function of membrane proteins often depends on the proteins' interaction with their lipid environment, spectacularly so in the case of mechanosensitive channels, which are gated through tension mediated by the surrounding lipids. Lipid bilayer tension is distributed quite inhomogeneously, but neither the scale at which relevant variation takes place nor the effect of varying lipid composition or tension has yet been investigated in atomic detail. We calculated lateral pressure profile distributions in lipid bilayers of various composition from all-atom molecular dynamics simulations totaling 110.5 ns in length. Reproducible pressure profile features at the 1 A length scale were determined. Lipids with phosphatidylcholine headgroups were found to shift the lateral pressure out of the hydrophobic core and into the headgroup region by an amount that is independent of area per lipid. POPE bilayers simulated at areas smaller than optimal exerted dramatically higher lateral pressure in a narrow region at the start of the aliphatic chain. Stretching of POPC bilayers increased tension predominantly in the same region. A simple geometric analysis for the gating of the mechanosensitive channel MscL suggests that pressure profiles affect its gating through the second moment of the profile in a tension-independent manner.     

Deformation free energy of bilayer membrane and its effect on gramicidin channel lifetime.

The deformation free energy of a lipid bilayer is presented based on the principle of a continuum theory. For small deformations, the free energy consists of a layer-compression term, a splay-distortion term, and a surface-tension term, equivalent to the elastic free energy of a two-layer smectic liquid crystal with surface tension. Minimization of the free energy leads to a differential equation that, with boundary conditions, determines the elastic deformation of a bilayer membrane. When a dimeric gramicidin channel is formed in a membrane of thickness greater than the length of the channel, the membrane deformation reduces the stability of the channel. Previously this effect was studied by comparing the variation of channel lifetime with the surface tension of bilayers (Elliott, J. R., D. Needham, J. P. Dilger, and D. A. Hayden, 1983, Biochim. Biophys. Acta, 735:95-103). The tension was assumed to pull a dimer for a distance z before the channel loses ion conductivity. To account for the data, z was found to be 18 A. With the deformation free energy, the data can be accounted for with z less than or approximately to 1 A, which is consistent with the breaking of hydrogen bonds in a dimer dissociation. Increasing the strength of lipid-protein interactions is not the only consequence of the complete free energy compared with the previous discussions. It also changes the shape of membrane deformation around an embedded channel from convex to concave, and increases the range of deformation from less than 10 A to greater than 20 A. Clearly these will be important factors in the general considerations of lipid-protein interactions and membrane-mediated interactions between proteins. In addition, thermal fluctuations of a membrane are calculated; in particular, we calculate the relations between the intrinsic thickness and the experimentally measured values. The experimental parameters of monoolein-squalene membranes are used for quantitative analyses.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating

In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation. Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature. Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches. Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state. Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate. We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.     

Correlation between the free energy of a channel-forming voltage-gated peptide and the spontaneous curvature of bilayer lipids

The aqueous-membrane partitioning of alamethicin, a voltage-gated channel-forming peptide, was measured as a function of the membrane spontaneous curvature. EPR spectroscopy was used to measure the partitioning of the peptide in lipid compositions formed from dioleoylphosphatidylcholine (DOPC) and varied percentages of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-N-methyl (DOPE-Me), or dioleoylphosphatidylethanolamine-N,N-dimethyl (DOPE-Me2). When the mole fraction of DOPE in mixtures of DOPC/DOPE is increased the binding of alamethicin decreases, and the increase in binding free energy is found to be linearly dependent upon the mole fraction of DOPE in the mixture. Addition of DOPE-Me or DOPE-Me2 also increases the binding free energy, except that the effect is reduced relative to that of DOPE. The free-energy increase per mole fraction of DOPE was found to be 1400 cal/mol, whereas for DOPE-Me and DOPE-Me2 the free-energy changes were 980 and 630 cal/mol, respectively. When the free-energy changes for alamethicin binding are compared with the previously determined spontaneous curvatures for mixtures of DOPC/DOPE and DOPC/DOPE-Me, the free energy of binding is found to be linearly dependent upon the spontaneous curvature of the bilayer lipids. The effects of membrane lipid unsaturation on the partitioning of alamethicin were also measured and are qualitatively consistent with this conclusion. The sensitivity to spontaneous curvature and the cooperativity that is seen in the binding curves for alamethicin are postulated to be a result of a localized thinning of the bilayer promoted by this peptide.     

Direct interaction of target SNAREs with the Kv2.1 channel. Modal regulation of channel activation and inactivation gating

Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.     

NMR-derived dynamic aspects of N-type inactivation of a Kv channel suggest a transient interaction with the T1 domain

Some eukaryotic voltage-gated K+ (Kv) channels contain an N-terminal inactivation peptide (IP), which mediates a fast inactivation process that limits channel function during membrane depolarization and thus shapes the action potential. We obtained sequence-specific nuclear magnetic resonance (NMR) assignments for the polypeptide backbone of a tetrameric N-terminal fragment (amino acids 1-181) of the Aplysia Kv1.1 channel. Additional NMR measurements show that the tetramerization domain 1 (T1) has the same globular structure in solution as previously determined by crystallography and that the IP (residues 1-20) and the linker (residues 21-65) are in a flexibly disordered, predominantly extended conformation. A potential contact site between the T1 domain and the flexible tail (residues 1-65) has been identified on the basis of chemical-shift changes of individual T1 domain amino acids, which map to the T1 surface near the interface between adjacent subunits. Paramagnetic perturbation experiments further indicate that, in the ensemble of solution conformers, there is at least a small population of species with the IP localized in close proximity to the proposed interacting residues of the T1 tetramer. Electrophysiological measurements show that all three mutations in this pocket that we tested slow the rate of inactivation and speed up recovery, as predicted from the preinactivation site model. These results suggest that specific, short-lived transient interactions between the T1 domain and the IP or the linker segment may play a role in defining the regulatory kinetics of fast channel inactivation.     

Solution structure of hpTX2, a toxin from Heteropoda venatoria spider that blocks Kv4.2 potassium channel

HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (L24). The high density in aromatic side chains of the putative functional surface as well as the lack of an asparagine is proposed to be the structural basis of the specificity of HpTX2 toward Kv4.2 channel.     

Hydration force and bilayer deformation: a reevaluation

The hydration repulsive force between lipid bilayers and the deformability of both gel and liquid-crystalline bilayers have been quantitated by an X-ray diffraction analysis of osmotically stressed liposomes. Both sampling theorem reconstructions and electron density distributions were calculated from diffraction data obtained from multilayers with applied osmotic pressures of 0-50 atm. The bilayer thickness and area per lipid molecule remain nearly constant (to within about 4%) in this pressure range, as adjacent bilayers move from their equilibrium separation in excess water to within 2-4 A of each other. This analysis indicates that the bilayers are relatively incompressible. This results differs from previously published X-ray diffraction studies of bilayer compressibility but agrees with direct mechanical measurements of the bilayer compressibility modulus. It is also found that the hydration repulsive force decays exponentially with separation between bilayers with a decay constant of 1.4 A for gel-state dipalmitoylphosphatidylcholine and 1.7 A for liquid-crystalline egg phosphatidylcholine bilayers. This implies that the exponential decay constant is not necessarily equal to the diameter of a water molecule, as has been previously suggested on experimental and theoretical grounds.     

Electrostatic interaction in the NH2-terminus accelerates inactivation of the Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a “ball and chain” inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH₂-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site.     

Constitutive activation of the Shaker Kv channel

In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521-532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.     

Measurement of histone-DNA interaction free energy in nucleosomes

Nucleosome positioning DNA sequences are of increasing interest because of their proposed roles in gene regulation and other chromosome functions in vivo, and because they have revealed new insights into the sequence-dependent structures and mechanics of DNA itself. Here, we describe methods to quantify the relative affinities of histone-DNA interactions in nucleosomes, i.e., the nucleosome positioning power of differing DNA sequences. We review methods developed by others and then discuss in detail our own approach to measurement of histone-DNA interaction free energies. Compared to earlier methods, our dialysis-based approach reduces the possibility that non-equilibrium or irreproducible results could be obtained. It facilitates a direct comparison of free energies for many sequences at the same time and it allows analysis of DNAs having a wide range of relative affinities.     

Adhesion energy of receptor-mediated interaction measured by elastic deformation

We investigated the role of receptor binding affinity in surface adhesion. A sensitive technique was developed to measure the surface energy of receptor-mediated adhesion. The experimental system involved a functionalized elastic agarose bead resting on a functionalized glass coverslip. Attractive intersurface forces pulled the two surfaces together, deforming the bead to produce an enlarged contact area. The Johnson-Kendall-Roberts (JKR) model was used to relate the surface energy of the interaction to the elasticity of the bead and the area of contact. The surface energies for different combinations of modified surfaces in solution were obtained from reflection interference contrast microscopy (RICM) measurements of the contact area formed by the bead and the coverslip. Studies with surfaces functionalized with ligand-receptor pairs showed that the relationship between surface energy and the association constant of the ligand binding has two regimes. At low binding affinity, surface energy increased linearly with the association constant, while surface energy increased logarithmically with the association constant in the high affinity regime.     

A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.     

A computer simulation of functional group contributions to free energy in water and a DPPC lipid bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.     

Surface free energy and interaction of Staphylococcus epidermidis with biomaterials

The adhesion of twenty nine Staphylococcus epidermidis strains to teflon, polyethylene, polycarbonate and bovine pericardium was studied in vitro and examined in relation to the surface free energies of both bacteria and biomaterials. All S. epidermidis strains had similar surface free energies, close to that of water, and adhered better to the materials with analogous surface free energies. There was a significant correlation (Kendall's Tau B = 1000) of biomaterial's surface free energy with the number of adhering bacteria. This correlation is inverse (Kendall's Tau B = -1000) when surface hydrophobicity is considered instead of surface free energy. This indicates that in Staphylococcus epidermidis adherence to biomaterials is inversely correlated to the surface hydrophobicity of the last, being so just the opposite of that occurring with other bacteria.     

Electrostatic interaction between inactivation ball and T1-S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a "ball and chain" inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the "ball and chain" inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1-S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1-S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.     

Mutating a critical lysine in ShK toxin alters its binding configuration in the pore-vestibule region of the voltage-gated potassium channel,Kv1.3

The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.     

Kv beta subunit oxidoreductase activity and Kv1 potassium channel trafficking.

Voltage-gated Kv1 potassium channels consist of pore-forming alpha subunits and cytoplasmic Kv beta subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv beta2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP(+) cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv beta subunit (Kv beta 1.1, Kv beta 1.2, Kv beta 2, and Kv beta 3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv beta subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv beta subunits promote cell surface expression of coexpressed Kv1.2 alpha subunits in transfected COS-1 cells. Kv beta1.1 and Kv beta 2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP(+) binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv beta subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP(+) binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv beta subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.