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The interaction between the amino terminus of Kv1‐type potassium channels and α‐actinin‐2 has been investigated. Using a combination of yeast two‐hybrid analysis and in vitro binding assays, α‐actinin‐2 was found to bind to the N‐termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3. Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel. The Kv1.5 binding sites in α‐actinin‐2 were found to lie within actinin's internal spectrin repeats. Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.

The interaction between the amino terminus of Kv1-type potassium channels and α-actinin-2 has been investigated. Using a combination of yeast two-hybrid analysis and in vitro binding assays, α-actinin-2 was found to bind to the N-termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3. Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel. The Kv1.5 binding sites in α-actinin-2 were found to lie within actinin’s internal spectrin repeats. Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C‐terminal region of IRK1 plays a crucial role in Mg2+i blockade and single‐channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time‐dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C‐terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single‐channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.

Dofetilide, a methanesulfonanilide derivative, is a potent class III antiarrhythmic drug. Like other members of this class of K+ channel blockers, the sites in the channel to which the drug binds are unknown, although high and low affinity binding has been reported in cardiomyocytes. The most sensitive K+ channel target for dofetilide seems to be IKr, the rapid component of the repolarizing delayed rectifier K+ current. However, block of other K+ channels occurs at higher concentrations and is of special interest in regard to toxicity. Recently, we have demonstrated that hIRK, a cloned inward rectifier K+ channel (IRK) isolated from human atrium and expressed heterologously in Xenopus oocytes, is blocked by dofetilide. We report the localization of a site that is critical for dofetilide block in hIRK. We used chimeric constructs between hIRK and ROMK1, a related inward rectifier that is drug resistant. Substitution of hIRK-M2, the second putative transmembrane spanning segment of IRKs, with ROMK1-M2 increased unblocking of dofetilide by 10-20-fold in hIRK. Site-directed mutagenesis further pinpointed the effects to a single hydrophobic residue (I177) in M2. A reduction in hydrophobicity by the point mutation I177C increased recovery from block > 10-fold (1.17 sec in wild-type to 0.112 sec at -80 mV at physiological K+ concentrations), leading us to suggest that hydrophobic interactions are essential for dofetilide block in hIRK. A similar mechanism may explain dofetilide block in other ion channels, including IKr.

Use of the antihistamine terfenadine has been associated with QT prolongation and torsade de pointes. One possible mechanism is blockade of cardiac potassium channels. We therefore characterized the effects of terfenadine on potassium currents recorded from isolated human cardiac myocytes. We demonstrated terfenadine block of the transient outward current and a novel, ultra-rapidly activating, delayed rectifier K+ current (IKur), which is very sensitive to 4-aminopyridine. IKur is probably produced by the protein product of Kv1.5a, a Shaker-like potassium channel cDNA cloned from human heart. We also compared terfenadine blockade of fHK (Kv1.5a) currents stably expressed in a human embryonic kidney cell line with terfenadine blockade of IKur in human atrial myocytes. Using the patch-clamp technique, we found that terfenadine produced a time-dependent reduction in Kv1.5a current that was consistent with blockade from the cytoplasmic side of the channel. The terfenadine-sensitive Kv1.5a current in human embryonic kidney cells was similar to the 4-aminopyridine-sensitive current in human atrial myocytes. In addition to blockade of the transient outward current and IKur, terfenadine at clinically relevant concentrations blocked both the rapidly and slowly activating components of the delayed rectifier in human atrial myocytes. Blockade of these K+ currents may contribute to the cardiotoxicity associated with terfenadine usage.

Kvbeta subunits have been shown to affect kinetic properties of voltage-gated K+ channel Kv1alpha subunits and increase the number of cell surface dendrotoxin-binding sites when coexpressed with Kv1. 2. Here, we show that Kvbeta1.2 alters both current expression and gating of Kvalpha1 channels and that each effect is mediated by a distinct Kvbeta1.2 domain. The Kvbeta1.2 N terminus or Kvalpha1-blocking domain introduced steady state current block, an apparent negative shift in steady state activation, and a slowing of deactivation along with a dramatic reduction in single channel open probability. N-terminal deletions of Kvbeta1.2 no longer altered channel kinetics but promoted dramatic increases in Kv1.2 current. The conserved Kvbeta1 C terminus or Kvalpha1 expression domain alone was sufficient to increase the number of functional channels. The same effect was observed with the normally noninactivating subunit, Kvbeta2. By contrast, Kv1.5 currents were reduced when coexpressed with either the Kvbeta1 C terminus or Kvbeta2, indicating that the Kvalpha1 expression domain has Kvalpha1 isoform-specific effects. Our results demonstrate that Kvbeta subunits consist of two domains that are separable on the basis of both primary structure and functional modulation of voltage-gated K+ channels.

1The Kvβ subunits of voltage‐gated K+channels alter the functional expression and gating of non‐ or slowly inactivating Kvα1 subunits via two separate domains. To determine how Kvβ subunits modulate a rapidly inactivating Kvα1 subunit, we did two‐microelectrode voltage clamp experiments on human Kv1.4 voltage‐gated K+channels expressed heterologously in Xenopusoocytes. In addition we tested a slowly inactivating mutant of Kv1.4 lacking amino acids 2–146 of the N‐terminal α‐ball domain (Kv1.4ΔN2‐146). Kv1.4 or Kv1.4ΔN2‐146 were co‐expressed with either rat Kvβ2 or human Kvβ1.2. To separate domain effects, we also used a mutant of Kvβ1.2 lacking the unique 79 amino acid N‐terminal β‐ball domain (Kvβ1‐C).2For the mutant Kv1.4ΔN2‐146 we found that Kvβ1‐C or Kvβ2 increased current amplitude without altering activation or inactivation. By contrast Kvβ1.2 produced rapid inactivation and slowed deactivation due to block produced by the β‐ball. The β‐ball also increased the rate of C‐type inactivation in 5 mm, but not 50 mm, external K+consistent with an effect of blockade on K+efflux.3For Kv1.4, Kvβ1‐C produced a voltage‐independent increase in the rate of inactivation and shifted the inactivation curve to more hyperpolarized potentials, but had no effect on deactivation. Kvβ1‐C, Kvβ2 and Kvβ1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the β‐ball. Kvβ1.2 produced an additional more rapid, voltage‐dependent component of inactivation, significantly reduced peak outward current and shifted steady‐state inactivation towards hyperpolarized potentials.4Yeast two‐hybrid studies showed that α‐β interaction was restricted to the N‐terminus of Kv1.4 and the C‐terminus of Kvβ1.2 or Kvβ2. Direct interaction with the α‐ball did not occur. Our interpretation is that Kvβ1‐C and Kvβ2 enhanced N‐type inactivation produced by the Kv1.4 α‐ball allosterically.5We propose that Kvβ1.2 has three effects on Kv1.4, the first two of which it shares with Kvβ2. First, Kvβ1‐C and Kvβ2 have a current‐enhancing effect. Second, Kvβ1‐C and Kvβ2 increase block by the α‐ball allosterically. Third, the β‐ball of Kβ1.2 directly blocks both Kv1.4 and Kv1.4ΔN2‐146. When both α‐ and β‐balls are present, competition for their respective binding sites slows the block produced by either ball.

Voltage-gated K+ (Kv) channels are important in the physiology of both excitable and nonexcitable cells. The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes. Given the fundamental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly, and metabolism. To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins. Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K+ channel-associated protein), which modulates the expression of Kv2 channels in heterologous expression system assays. KChAP interacts with the N termini of Kvalpha2 subunits, as well as the N termini of Kvalpha1 and the C termini of Kvbeta subunits. Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins. The amplitudes of Kv2. 1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected. Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents. We suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels.

Both in vivo and in vitro, neurofilaments (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofilament-specific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affinity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of greater than 90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone H1 kinase. The molecular properties and specific sequence requirements of the NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hyperphosphorylated.

Voltage-gated K+ (Kv) channels consist of alpha subunits complexed with cytoplasmic Kvbeta subunits. Kvbeta1 subunits enhance the inactivation of currents expressed by the Kv1 alpha subunit subfamily. Binding has been demonstrated between the C terminus of Kvbeta1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker alpha subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvbeta subunits, 1.2 and 1.3, with Kvalpha subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvbeta subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvalpha subunit. In functional experiments, we found differences in modulation of Kv1alpha subunit currents that we attribute to the unique N-terminal domains of the two Kvbeta subunits. Both Kvbeta subunits act as open channel blockers at physiological membrane potentials, but hKvbeta1.2 is a more potent blocker than hKvbeta1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1. 5. Moreover, hKvbeta1.2 is sensitive to redox conditions, whereas hKvbeta1.3 is not. We suggest that different Kvbeta subunits extend the range over which distinct Kv1alpha subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C‐terminal region of IRK1 plays a crucial role in Mg2+i blockade and single‐channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time‐dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C‐terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single‐channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.

Perhexiline maleate is an antianginal drug that has been shown to have antiarrhythmic effects in humans. To examine whether some of these clinical observations could be caused by block of cardiac K+ channels, we examined the effects of perhexiline on a rapidly activating delayed rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in human embryonic kidney cells as well as a corresponding K+ current (the ultra-rapid delayed rectifier, IKur) in human atrial myocytes. With the use of inside-out macropatches, we found that perhexiline inhibited Kv1.5 current in a time- and voltage-dependent manner with an IC50 value of 1.5 x 10(-6) M at +50 mV. Perhexiline reduced Kv1.5 tail current amplitude and slowed its decay relative to control. These data are consistent with blockade of open channels, probably from the intracellular surface. Perhexiline (3 microM) also blocked IKur in human atrial myocytes. The block that was observed was both time- and voltage-dependent in qualitatively similar ways to block of Kv1.5 channels. However, the time-dependent block of IKur by perhexiline was somewhat slower and its voltage-dependence steeper relative to its effects on Kv1.5. These data indicate that perhexiline blocks both cloned and native human cardiac K+ channels. Blockade of one or more types of voltage-dependent K+ channels may explain some of the electrophysiological effects of perhexiline observed in humans.

Inwardly rectifying K+ channels (IRKs) are highly K(+)-selective, integral membrane proteins that help maintain resting the membrane potential and cell volume. Integral membrane proteins as a class are frequently N-glycosylated with the attached carbohydrate being extracellular and perhaps modulating function. However, dynamic effects of glycosylation have yet to be demonstrated at the molecular level. ROMK1, a member of the IRK family is particularly suited to the study of glycosylation because it has a single N-glycosylation consensus sequence (Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Herbert, S. C. (1993) Nature 362, 31-38). We show that ROMK1 is expressed in a functional state in the plasmalemma of an insect cell line (Spodoptera frugiperda, Sf9) and has two structures, glycosylated and unglycosylated. To test functionality, glycosylation was abolished by an N117Q mutation or by treatment with tunicamycin. Whole cell currents were greatly reduced in both of the unglycosylated forms compared to wild-type. Single channel currents revealed a dramatic decrease in opening probability, po, as the causative factor. Thus we have shown biochemically that the N-glycosylation sequon is extracellular, a result consistent with present topological models of IRKs, and we conclude that sequon occupancy by carbohydrate stabilizes the open state of ROMK1.

Verapamil is an antagonist of L-type Ca2+ channels, and part of its binding site is located in the sixth transmembrane segment (S6) in the fourth repeat of the protein. Verapamil also blocks K+ channels, which are members of the same supergene family as Ca2+ channels. We examined the effects of verapamil on a rapidly activating delayed rectifier K+ channel (designated fHK) cloned from human heart. Verapamil inhibited 86Rb+ efflux from fHK-transfected human embryonic kidney cells with an EC50 of 4.5 x 10(-5) M. Whole-cell patch-clamp experiments revealed that verapamil induced a rapid component of fHK current inactivation but was without effect on activation. The effect was concentration and voltage dependent and was attributed to open channel blockade. The apparent association and dissociation rate constants measured at +50 mV were about 1.65 x 10(5) M-1 sec-1 and 3.48 sec-1, respectively. S6 of fHK has significant homology to that portion of the verapamil binding site identified in Ca2+ channels, and S6 is thought to form part of the inner mouth of K+ channel pores. The data support a role for verapamil as a blocker of the inner pore of voltage-dependent K+ channels in human myocardium.

Use of the nonsedating antihistamine terfenadine has been associated with altered cardiac repolarization in certain clinical settings. For this reason we examined the effects of terfenadine, and its metabolites, on a rapidly activating delayed rectifier K+ channel (fHK) cloned from human heart. fHK was stably expressed in human embryonic kidney cells, and both whole-cell currents and currents from excised inside-out patches were recorded. Terfenadine (3 microM) blocked whole-cell fHK current by 72 +/- 6%. In inside-out patches, terfenadine applied to the cytoplasmic surface blocked fHK with an IC50 value of 367 nM. The main effect of terfenadine was to enhance the rate of inactivation of fHK current and thereby reduce the current at the end of a prolonged voltage-clamp pulse. The blockade displayed a weak voltage dependence, increasing at more positive potentials. The mechanism of action of terfenadine is therefore consistent with blockade of open channels. In contrast, the metabolites of terfenadine were weakly active on fHK. IC50 values for all of the metabolites tested ranged from 27-fold to 583-fold higher than that obtained for terfenadine. It is concluded that terfenadine, but not its metabolites, blocks at least one type of human cardiac K+ channel at clinically relevant concentrations and that this activity may underlie the cardiac arrhythmias that have been associated with the use of this drug.