Donald L. Campbell

Transient outward potassium current, 'Ito', phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms.

At least two functionally distinct transient outward K+ current (Ito) phenotypes can exist across the free wall of the left ventricle (LV). Based upon their voltage‐dependent kinetics of recovery from inactivation, these two phenotypes are designated ‘Ito,fast’ (recovery time constants on the order of tens of milliseconds) and ‘Ito,slow’ (recovery time constants on the order of thousands of milliseconds). Depending upon species, either Ito,fast, Ito,slow or both current phenotypes may be expressed in the LV free wall. The expression gradients of these two Ito phenotypes across the LV free wall are typically heterogeneous and, depending upon species, may consist of functional phenotypic gradients of both Ito,fast and Ito,slow and/or density gradients of either phenotype. We review the present evidence (molecular, biophysical, electrophysiological and pharmacological) for Kv4.2/4.3 α subunits underlying LV Ito,fast and Kv1.4 α subunits underlying LV Ito,slow and speculate upon the potential roles of each of these currents in determining frequency‐dependent action potential characteristics of LV subepicardial versus subendocardial myocytes in different species. We also review the possible functional implications of (i) ancillary subunits that regulate Kv1.4 and Kv4.2/4.3 (Kvβ subunits, DPPs), (ii) KChIP2 isoforms, (iii) spider toxin‐mediated block of Kv4.2/4.3 (Heteropoda toxins, phrixotoxins), and (iv) potential mechanisms of modulation of Ito,fast and Ito,slow by cellular redox state, [Ca2+]i and kinase‐mediated phosphorylation. Ito phenotypic activation and state‐dependent gating models and molecular structure–function relationships are also discussed.

In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes

The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.

C-type inactivation controls recovery in a fast inactivating cardiac K+ channel (Kv1.4) expressed in Xenopus oocytes.

1. A fast inactivating transient K+ current (FK1) cloned from ferret ventricle and expressed in Xenopus oocytes was studied using the two‐electrode voltage clamp technique. Removal of the NH2‐terminal domain of FK1 (FK1 delta 2‐146) removed fast inactivation consistent with previous findings in Kv1.4 channels. The NH2‐terminal deletion mutation revealed a slow inactivation process, which matches the criteria for C‐type inactivation described for Shaker B channels. 2. Inactivation of FK1 delta 2‐146 at depolarized potentials was well described by a single exponential process with a voltage‐insensitive time constant. In the range ‐90 to +20 mV, steady‐state C‐type inactivation was well described by a Boltzmann relationship that compares closely with inactivation measured in the presence of the NH2‐terminus. These results suggest that C‐type inactivation is coupled to activation. 3. The coupling of C‐type inactivation to activation was assessed by mutation of the fourth positively charged residue (arginine 454) in the S4 voltage sensor to glutamine (R454Q). This mutation produced a hyperpolarizing shift in the inactivation relationship of both FK1 and FK1 delta 2‐146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1 delta 2‐146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]o and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1 delta 2‐146. The similarity in recovery rates in response to these perturbations suggests that recovery from C‐type inactivation governs the overall rate of recovery of inactivated channels for both FK1 and FK1 delta 2‐146. 6. Analysis of the rate of recovery of FK1 channels for inactivating pulses of different durations (70‐2000 ms) indicates that recovery rate is insensitive to the duration of the inactivating pulse.

Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart Implications for Mechanisms Governing Indirect and Direct Nitric Oxide–Related Effects

The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO*/O(2-) interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.

Heterogeneous expression of KChIP2 isoforms in the ferret heart

Kv4 channels are believed to underlie the rapidly recovering cardiac transient outward current (Ito) phenotype. However, heterologously expressed Kv4 channels fail to fully reconstitute the native current. Kv channel interacting proteins (KChIPs) have been shown to modulate Kv4 channel function. To determine the potential involvement of KChIPs in the rapidly recovering Ito, we cloned three KChIP2 isoforms (designated fKChIP2, 2a and 2b) from the ferret heart. Based upon immunoblot data suggesting the presence of a potential endogenous KChIP‐like protein in HEK 293, CHO and COS cells but absence in Xenopus oocytes, we coexpressed Kv4.3 and the fKChIP2 isoforms in Xenopus oocytes. Functional analysis showed that while all fKChIP2 isoforms produced a fourfold acceleration of recovery kinetics compared to Kv4.3 expressed alone, only fKChIP2a produced large depolarizing shifts in the V1/2 of steady‐state activation and inactivation as seen for the native rapidly recovering Ito. Analysis of RNA and protein expression of the three fKChIP2 isoforms in ferret ventricles showed that fKChIP2b was most abundant and was expressed in a gradient paralleling the rapidly recovering Ito distribution. Ferret KChIP2 and 2a were expressed at very low levels. The ventricular expression distribution suggests that fKChIP2 isoforms are involved in modulation of the rapidly recovering Ito; however, additional regulatory factors are also likely to be involved in generating the native current.

Elucidating KChIP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChIP2 isoform.

Kv channel interacting proteins (KChIPs) are Ca2+‐binding proteins with four EF‐hands. KChIPs modulate Kv4 channel gating by slowing inactivation kinetics and accelerating recovery kinetics. Thus, KChIPs are believed to be important regulators of Kv4 channels underlying transient outward K+ currents in many excitable cell types. We have cloned a structurally minimal KChIP2 isoform (KChIP2d) from ferret heart. KChIP2d corresponds to the final 70 C‐terminal amino acids of other KChIPs and has only one EF‐hand. We demonstrate that KChIP2d is a functional KChIP that both accelerates recovery and slows inactivation kinetics of Kv4.3, indicating that the minimal C‐terminus can maintain KChIP regulatory properties. We utilize KChIP2d to further demonstrate that: (i) the EF‐hand modulates effects on Kv4.3 inactivation but not recovery; (ii) Ca2+‐dependent effects on Kv4.3 inactivation are mediated through a mechanism reflected in the slow time constant of inactivation; and (iii) a short stretch of amino acids exclusive of the EF‐hand partially mediates Ca2+‐independent effects on recovery. Our results demonstrate that distinct regions of a KChIP molecule are involved in modulating inactivation and recovery. The potential ability of KChIP EF‐hands to sense intracellular Ca2+ levels and transduce these changes to alterations in Kv4 channel inactivation kinetics may serve as a mechanism allowing intracellular Ca2+ transients to modulate repolarization. KChIP2d is a valuable tool for elucidating structural domains of KChIPs involved in Kv4 channel regulation.

Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes.

Ferret atrial myocytes can display an E-4031-sensitive current (IKr) that is similar to that previously described for guinea pig cardiac myocytes. We examined the ferret atrial IKr as the E-4031-sensitive component of current using the amphotericin B perforated patch-clamp technique. Steady-state IKr during depolarizing pulses showed characteristic inward rectification. Activation time constants during a single pulse were voltage dependent, consistent with previous studies. However, for potentials positive to +30 mV, IKr time course became complex and included a brief transient component. We examined the envelope of tails of the drug-sensitive current for activation in the range -10 to +50 mV and found that the tail currents for IKr do not activate with the same time course as the current during the depolarizing pulse. The activation time course determined from tail currents was relatively voltage insensitive over the range +30 to +50 mV (n = 5), but was voltage sensitive for potentials between -10 and +30 mV and appeared to show some sigmoidicity in this range. These data indicate that activation of IKr occurs in at least two steps, one voltage sensitive and one voltage insensitive, the latter of which becomes rate limiting at positive potentials. We also examined the rapid time-dependent inactivation process that mediates rectification at positive potentials. The time constants for this process were only weakly voltage dependent over the range of potentials from -50 to +60 mV. From these data we constructed a simple linear four-state model that reproduces the general features of ferret IKr, including the initial transient at positive potentials and the apparent discrepancy between the currents during the initial depolarizing pulse and the tail current.

Regulation of Kv4.3 voltage-dependent gating kinetics by KChIP2 isoforms

We conducted a kinetic analysis of the voltage dependence of macroscopic inactivation (τfast, τslow), closed‐state inactivation (τclosed,inact), recovery (τrec), activation (τact), and deactivation (τdeact) of Kv4.3 channels expressed alone in Xenopus oocytes and in the presence of the calcium‐binding ancillary subunits KChIP2b and KChIP2d. We demonstrate that for all expression conditions, τrec, τclosed,inact and τfast are components of closed‐state inactivation transitions. The values of τclosed,inact and τfast monotonically merge from −30 to −20 mV while the values of τclosed,inact and τrec approach each other from −60 to −50 mV. These data generate classic bell‐shaped time‐constant–potential curves. With the KChIPs, these curves are distinct from that of Kv4.3 expressed alone due to acceleration of τrec and slowing of τclosed,inact and τfast. Only at depolarized potentials where channels open is τslow detectable suggesting that it represents an open‐state inactivation mechanism. With increasing depolarization, KChIPs favour this open‐state inactivation mechanism, supported by the observation of larger transient reopening currents upon membrane hyperpolarization compared to Kv4.3 expressed alone. We propose a Kv4.3 gating model wherein KChIP2 isoforms accelerate recovery, slow closed‐state inactivation, and promote open‐state inactivation. This model supports the observations that with KChIPs, closed‐state inactivation transitions are [Ca2+]i‐independent, while open‐state inactivation is [Ca2+]i‐dependent. The selective KChIP‐ and Ca2+‐dependent modulation of Kv4.3 inactivation mechanisms predicted by this model provides a basis for dynamic modulation of the native cardiac transient outward current by intracellular Ca2+ fluxes during the action potential.

Modulation of L-type Ca2+ current by extracellular ATP in ferret isolated right ventricular myocytes.

1. The effects of extracellular adenosine triphosphate (ATP) on the basal L‐type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole‐cell and cell‐attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration‐dependent manner, without any significant changes in the voltage dependence of either the peak ICa I‐V relationship or steady‐state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady‐state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at ‐70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8‐cyclopentyl‐3,4‐dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2‐methyl‐thio‐ATP > ATP > alpha,beta‐methylene‐ATP, indicating involvement of a P2Y‐type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP‐induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX‐insensitive G protein in the P2Y receptor‐coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect was not obligatorily dependent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble‐average current behaviour constructed from cell‐attached patch recordings of single L‐type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 microM ATP was added to the superfusate on the outside of the patch electrode the inhibition of ICa was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity in K+ Channel Transcript Expression Detected in Isolated Ferret Cardiac Myocytes

The molecular basis of the potassium ion (K+) channels that generate repolarization in heart tissue remains uncertain, in part because of the molecular diversity of the voltage‐gated K+ channel family. In our investigation, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes to determine the identity, regional distribution, and cellular distribution of voltage‐gated K+ channel, a‐subunit mRNA expressed in ferret heart. The regions studied were from the sinoatrial node (SA), right and left atrium, right and left ventricle, and interatrial and interventricular septa, Kv1.5 and Kv1.4 were the most widely distributed K+ channel transcripts in the ferret heart (present in approximately 70%–86% and approximately 46%–95% of tested myocytes, respectively), followed byKv1.2, Kv2.1, and Kv4.2. In addition, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family, Kv1.1, Kv1.6, and Kv6.1 were rarely expressed in working myocytes, but were more commonly expressed in SA nodal cells. Two other transcripts whose genes have been implicated in the long QT syndrome, erg and KvLQT1, were common in all regions (approximately 41%–58% and 52%–72%, respectively). These results show that both the diversity and heterogeneity of K+ channel mRNA in heart tissue is greater than previously suspected