David F. Steele

α‐Actinin‐2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells

Voltage‐gated K+ (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD‐95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through α‐actinin‐2 to the post‐synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439–442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with α‐actinin‐2. Human Kv1.5 interacts via its N‐terminus/core region and can be immunoprecipitated with α‐actinin‐2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and α‐actinin‐2 co‐localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to α‐actinin‐2 modulate the ion and gating current density.

Kv1.5 Is an Important Component of Repolarizing K+ Current in Canine Atrial Myocytes

Abstract— Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.

Kv1.5 Surface Expression Is Modulated by Retrograde Trafficking of Newly Endocytosed Channels by the Dynein Motor

In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein–dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57±0.07 nA/pF, n=12; to 1.18±0.2 nA/pF, n=12, P<0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K+ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K+ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated.

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of α‐actinin‐2

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 actinins 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.

Mechanisms of cardiac potassium channel trafficking

The regulation of ion channels involves more than just modulation of their synthesis and kinetics, as controls on their trafficking and localization are also important. Although the body of knowledge is fairly large, the entire trafficking pathway is not known for any one channel. This review summarizes current knowledge on the trafficking of potassium channels that are expressed in the heart. Our knowledge of channel assembly, trafficking through the Golgi apparatus and on to the surface is covered, as are controls on channel surface retention and endocytosis.

SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism

The functional interaction of the voltage‐gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated. In marked contrast with the known dependence of SAP97‐induced Kv1 potassium current down‐regulation on the channel C‐termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N‐terminus. Deletion of the Kv1.5 N‐terminus eliminated the SAP97‐mediated increase in potassium currents whereas deletion of the channels C‐terminal PDZ binding motif had no effect. In contrast with other Kv1–SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins. The proteins did not co‐localize in cardiac myocytes nor did they co‐immunoprecipitate from transfected HEK cells. Yeast two‐hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co‐immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes. Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.

Amino-terminal determinants of U-type inactivation of voltage-gated K+ channels

The T1 domain is a cytosolic NH2-terminal domain present in all Kv (voltage-dependent potassium) channels, and is highly conserved between Kv channel subfamilies. Our characterization of a truncated form of Kv1.5 (Kv1.5ΔN209) expressed in myocardium demonstrated that deletion of the NH2 terminus of Kv1.5 imparts a U-shaped inactivation-voltage relationship to the channel, and prompted us to investigate the NH2 terminus as a regulatory site for slow inactivation of Kv channels. We examined the macroscopic inactivation properties of several NH2-terminal deletion mutants of Kv1.5 expressed in HEK 293 cells, demonstrating that deletion of residues up to the T1 boundary (Kv1.5ΔN19, Kv1.5ΔN91, and Kv1.5ΔN119) did not alter Kv1.5 inactivation, however, deletion mutants that disrupted the T1 structure consistently exhibited inactivation phenotypes resembling Kv1.5ΔN209. Chimeric constructs between Kv1.5 and the NH2 termini of Kv1.1 and Kv1.3 preserved the inactivation kinetics observed in full-length Kv1.5, again suggesting that the Kv1 T1 domain influences slow inactivation. Furthermore, disruption of intersubunit T1 contacts by mutation of residues Glu131 and Thr132to alanines resulted in channels exhibiting a U-shaped inactivation-voltage relationship. Fusion of the NH2terminus of Kv2.1 to the transmembrane segments of Kv1.5 imparted a U-shaped inactivation-voltage relationship to Kv1.5, whereas fusion of the NH2 terminus of Kv1.5 to the transmembrane core of Kv2.1 decelerated Kv2.1 inactivation and abolished the U-shaped voltage dependence of inactivation normally observed in Kv2.1. These data suggest that intersubunit T1 domain interactions influence U-type inactivation in Kv1 channels, and suggest a generalized influence of the T1 domain on U-type inactivation between Kv channel subfamilies.

Internalized Kv1.5 traffics via Rab-dependent pathways

Little is known about the postinternalization trafficking of surface‐expressed voltage‐gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage‐gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5‐and Rab4‐positive endosomes, suggesting that the channel is internalized via a Rab5‐dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 ± 213 pA pF−1 from control 675 ± 81.6 pA pF−1. Rab4DN similarly increased Kv1.5 currents to 1382 ± 155 pA pF−1 from the control 522 ± 82.7 pA pF−1 at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5‐fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over‐expression of wild‐type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 ± 96.3 pA pF−1 from the control 869 ± 135.5 pA pF−1. The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule‐dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.

N‐terminal PDZ‐binding domain in Kv1 potassium channels

We have investigated the interactions of prototypical PDZ domains with both the C‐ and N‐termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two‐hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C‐ and N‐termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co‐immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C‐terminal PDZ‐binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co‐localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ‐binding motif. Deletion analysis localized the N‐terminal PDZ‐binding site in Kv1.5 to the T1 region of the channel. Co‐expression of PSD95 with Kv1.5 N‐ and C‐terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.