Jodene Eldstrom

Heterogeneous expression of repolarizing, voltage‐gated K+ currents in adult mouse ventricles

Previous studies have documented the expression of four kinetically distinct voltage‐gated K+ (Kv) currents, Ito,f, Ito,s, IK,slow and Iss, in mouse ventricular myocytes and demonstrated that Ito,f and Ito,s are differentially expressed in the left ventricular apex and the interventricular septum. The experiments here were undertaken to test the hypothesis that there are further regional differences in the expression of Kv currents or the Kv subunits (Kv4.2, Kv4.3, KChIP2, Kv1.5, Kv2.1) encoding these currents in adult male and female (C57BL6) mouse ventricles. Whole‐cell voltage‐clamp recordings revealed that mean (±s.e.m.) peak outward K+ current and Ito,f densities are significantly (P < 0.001) higher in cells isolated from the right (RV) than the left (LV) ventricles. Within the LV, peak outward K+ current and Ito,f densities are significantly (P < 0.05) higher in cells from the apex than the base. In addition, Ito,f and IK,slow densities are lower in cells isolated from the endocardial (Endo) than the epicardial (Epi) surface of the LV wall. Importantly, similar to LV apex cells, Ito,s is not detected in RV, LV base, LV Epi or LV Endo myocytes. No measurable differences in K+ current densities or properties are evident in RV or LV cells from adult male and female mice, although Ito,f, Ito,s, IK,slow and Iss densities are significantly (P < 0.01) higher, and action potential durations at 50% (APD50) are significantly (P < 0.05) shorter in male septum cells. Western blot analysis revealed that the expression levels of Kv4.2, Kv4.3, KChIP2, Kv1.5 and Kv2.1 are similar in male and female ventricles. In addition, consistent with the similarities in repolarizing Kv current densities, no measurable differences in ECG parameters, including corrected QT (QTc) intervals, are detected in telemetric recordings from adult male and female (C57BL6) mice.

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.

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.

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current IKur. Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MβCD induced a delayed (≈7 min) increase in IKur; the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MβCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MβCD-induced IKur increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MβCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.

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.

The molecular basis of high-affinity binding of the antiarrhythmic compound vernakalant (RSD1235) to Kv1.5 channels.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355–2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.

Localization of Kv1.5 channels in rat and canine myocyte sarcolemma

Voltage‐gated potassium (Kv) channel subtypes localize to the plasma membrane of a number of cell types, and the sarcolemma in myocytes. Because many signaling molecules concentrate in subdomains of the plasma membrane, the localization of Kv channels to these sites may have important implications for channel function and regulation. In this study, the association of the voltage‐gated potassium channel Kv1.5 with a specific subtype of lipid rafts, caveolae, in rat and canine cardiac myocytes has been investigated. Interactions between caveolin‐3 and β‐dystroglycan or eNOS, as well as between Kv1.5 and α‐actinin were readily detected in co‐immunoprecipitation experiments, whereas no association between Kv1.5 and caveolin‐3 was evident. Wide‐field microscopy and deconvolution techniques revealed that the percent co‐localization of Kv1.5 with caveolin‐3 was extremely low in atrial myocytes from rat and canine hearts (8 ± 1% and 12.2 ± 2%, respectively), and limited in ventricular myocytes (11 ± 4% and 20 ± 3% in rat and canine, respectively). Immunoelectron microscopic imaging of rat atrial and ventricular tissues showed that Kv1.5 and caveolin‐3 labeling generally did not overlap. In HEK293 cells stably expressing the channel, Kv1.5 did not target to the low buoyant density raft fraction along with flotillin but instead fractionated along with the non‐raft associated transferrin receptor. Taken together, these results suggest that Kv1.5 is not present in caveolae of rat and canine heart.

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.

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.

Single-channel basis for the slow activation of the repolarizing cardiac potassium current, IKs

Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel IKs. Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel IKs. Single-channel recordings from transiently transfected mouse ltk− cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type IKs channel required modification of existing Markov models to include these features of channel behavior.