KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression.

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4.

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4.

Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome.

Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG (KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K(+) currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.

KChAP as a chaperone for specific K(+) channels.

The concept of chaperones for K(+) channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, I(to).

Separable effects of human Kvbeta1.2 N- and C-termini on inactivation and expression of human Kv1.4.

1. The Kvbeta subunits of voltage-gated K+ channels alter the functional expression and gating of non- or slowly inactivating Kvalpha1 subunits via two separate domains. To determine how Kvbeta subunits modulate a rapidly inactivating Kvalpha1 subunit, we did two-microelectrode voltage clamp experiments on human Kv1.4 voltage-gated K+ channels expressed heterologously in Xenopus oocytes. In addition we tested a slowly inactivating mutant of Kv1.4 lacking amino acids 2-146 of the N-terminal alpha-ball domain (Kv1. 4DeltaN2-146). Kv1.4 or Kv1.4DeltaN2-146 were co-expressed with either rat Kvbeta2 or human Kvbeta1.2. To separate domain effects, we also used a mutant of Kvbeta1.2 lacking the unique 79 amino acid N-terminal beta-ball domain (Kvbeta1-C). 2. For the mutant Kv1.4DeltaN2-146 we found that Kvbeta1-C or Kvbeta2 increased current amplitude without altering activation or inactivation. By contrast Kvbeta1.2 produced rapid inactivation and slowed deactivation due to block produced by the beta-ball. The beta-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. 3. For Kv1.4, Kvbeta1-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. Kvbeta1-C, Kvbeta2 and Kvbeta1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the beta-ball. Kvbeta1.2 produced an additional more rapid, voltage-dependent component of inactivation, significantly reduced peak outward current and shifted steady-state inactivation towards hyperpolarized potentials. 4. Yeast two-hybrid studies showed that alpha-beta interaction was restricted to the N-terminus of Kv1.4 and the C-terminus of Kvbeta1. 2 or Kvbeta2. Direct interaction with the alpha-ball did not occur. Our interpretation is that Kvbeta1-C and Kvbeta2 enhanced N-type inactivation produced by the Kv1.4 alpha-ball allosterically. 5. We propose that Kvbeta1.2 has three effects on Kv1.4, the first two of which it shares with Kvbeta2. First, Kvbeta1-C and Kvbeta2 have a current-enhancing effect. Second, Kvbeta1-C and Kvbeta2 increase block by the alpha-ball allosterically. Third, the beta-ball of Kbeta1.2 directly blocks both Kv1.4 and Kv1.4DeltaN2-146. When both alpha- and beta-balls are present, competition for their respective binding sites slows the block produced by either ball.

Cloning and expression of a novel K+ channel regulatory protein, KChAP.

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.

Separable Kvbeta subunit domains alter expression and gating of potassium channels.

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.

Interactions among inactivating and noninactivating Kvbeta subunits, and Kvalpha1.2, produce potassium currents with intermediate inactivation.

Experiments were carried out to determine whether coinjection of Kvalpha1.2 with inactivating and noninactivating Kvbeta subunits would produce currents with intermediate kinetics and channel complexes containing a mixture of these subunits. Upon coexpression with a saturating amount of Kvbeta1.2 and increasing levels of a noninactivating deletion mutant of Kvbeta1.2, we show that macroscopic Kvalpha1.2 currents have levels of fractional inactivation and inactivation time constants that are intermediate between those obtained with either the inactivating Kvbeta1.2 or the noninactivating Kvbeta1.2 mutant. We also find that coexpression of Kvalpha1.2 with saturating amounts of Kvbeta1.2 and the deletion mutant produces a population of single channels with properties intermediate to either the inactivating or noninactivating parental phenotype. Our data can best be explained by the presence of an intermediate population of heterooligomeric channels consisting of Kvalpha1.2 with different combinations of both types of subunits. Since Kvalpha1.2 subunits coexist in cells with inactivating and noninactivating Kvbeta subunits, our findings suggest that heterooligomeric assembly of these subunits occurs to increase the range of K+ current kinetics and expression levels.

Comparison of binding and block produced by alternatively spliced Kvbeta1 subunits.

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.

C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.

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.

Potassium channel structure and function as reported by a single glycosylation sequon.

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.

Cloning and functional expression of an inwardly rectifying K+ channel from human atrium.

The cardiac inward rectifier current (IK1) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K+ channels (IRKs) from different tissues have been cloned recently, the nature and number of K+ channels contributing to the cardiac IK1 are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase-polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited < 70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in Xenopus oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac IK1 in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.

Molecular cloning and functional expression of a novel potassium channel beta-subunit from human atrium.

We report the cloning and functional expression of a novel K+ channel beta-subunit from human atrium, hKv beta 3. hKv beta 3 is highly homologous to the two beta-subunits cloned from rat brain, Kv beta 1 and Kv beta 2, but has an essentially unique stretch of 79 N-terminal residues. Upon expression in Xenopus oocytes, hKv beta 3 accelerates the inactivation of co-injected hKv1.4 currents and induces fast inactivation of non-inactivating co-injected hKv1.5 currents. By contrast, hKv beta 3 had no effect on hKv1.1, hKv1.2, or hKv2.1 currents. Thus, hKv beta 3 represents a third type of K+ channel beta-subunit which modulates the kinetics of a unique subset of channels in the Kv1 subfamily.

Spermine and spermidine as gating molecules for inward rectifier K+ channels.

Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.

Gating of inwardly rectifying K+ channels localized to a single negatively charged residue.

Inwardly rectifying K+ channels (IRKs) conduct current preferentially in the inward direction. This inward rectification has two components: voltage-dependent blockade by intracellular Mg2+ (Mg2+i) and intrinsic gating. Two members of this channel family, IRK1 (ref. 10) and ROMK1 (ref. 11), differ markedly in affinity for Mg2+i (ref. 12). We found that IRK1 and ROMK1 differ in voltage-dependent gating and searched for the gating structure by large-scale and site-directed mutagenesis. We found that a single amino-acid change within the putative transmembrane domain M2, aspartate (D) in IRK1 to the corresponding asparagine (N) in ROMK1, controls the gating phenotype. Mutation D172N in IRK1 produced ROMK1-like gating whereas the reverse mutation in ROMK1--N171D--produced IRK1-like gating. Thus, a single negatively charged residue seems to be a crucial determinant of gating.

Specification of pore properties by the carboxyl terminus of inwardly rectifying K+ channels.

Inwardly rectifying potassium (K+) channels (IRKs) maintain the resting membrane potential of cells and permit prolonged depolarization, such as during the cardiac action potential. Inward rectification may result from block of the ion conduction pore by intracellular magnesium (Mgi2+). Two members of this family, IRK1 and ROMK1, which share 40 percent amino acid identity, differ markedly in single-channel K+ conductance and sensitivity to block by Mgi2+. The conserved H5 regions were hypothesized to determine these pore properties because they have this function in voltage-dependent K+ channels and in cyclic nucleotide-gated channels. However, exchange of the H5 region between IRK1 and ROMK1 had no effect on rectification and little or no effect on K+ conductance. By contrast, exchange of the amino- and carboxyl-terminal regions together transferred Mg2+ blockade and K+ conductance of IRK1 to ROMK1. Exchange of the carboxyl but not the amino terminus had a similar effect. Therefore, the carboxyl terminus appears to have a major role in specifying the pore properties of IRKs.

Stable expression and regulation of a rat brain K+ channel.

The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K+ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to Kv3.1 channels expressed transiently in Xenopus oocytes. Kv3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased Kv3.1 currents. Based on these results, we find that the stable expression of voltage-gated K+ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.

Resolution and purification of a neurofilament-specific kinase.

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.