Comprehensive analysis of two Shank3 and the Cacna1c mouse models of autism spectrum disorder.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng's Shank3tm2Gfng model, hereafter Shank3/F, Jiang's Shank3tm1Yhj model, hereafter Shank3/J and the Cacna1c deletion model. The Shank3 models mimick gene mutations associated with Phelan-McDermid Syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. This study utilizes both standard and novel behavioral tests with the same methodology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. We found that some but not all behaviors replicated published findings and those that did replicate, such as social behavior and overgrooming in Shank3 models, tended to be milder than reported elsewhere. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, only Shank3/F showed differences in sensory-gating. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

SU-E-T-197: A Comprehensive Variance Reporting System and an Analysis of Variances Reported at Our Institution.

PURPOSE: It is essential for radiation oncology departments to have comprehensive patient safety and quality programs. Two years ago we undertook a systematic review of our safety/QA program. Existing policies were updated and new policies created where necessary. One crucial component of any safety/QA program is continually updating it based on current information, the 'check' and 'act' portions of the Deming Cycle. We accomplished this with a transparent variance reporting system and a safety/QA committee reviewing and acting on reported variances. METHODS: With 5 radiation oncology centers in our institution, we needed to devise a system that would allow anyone to report a variance and provide our QA committee the ability to review variances system-wide. We developed the system using web-based tools. The system allows individuals to report variances, anonymously or named, specify the nature of the variance and indicate the tools used to identify the variance. RESULTS: In 2011, 285 variances were reported, 102 were reported by physicists, 86 anonymously, 71 by therapists and 26 by dosimetrists. We realized the need to develop clear classifications for variances. We added a high priority category, defined as variances which resulted in or had the potential to result in harm to a patient or when a policy is purposely overridden. Of the 285 variances reported, 5 were high priority. We created a process variance category, defined as variances where a specific clinical process is not followed. Of the 285 reported variances 155 were process variances. CONCLUSIONS: Reporting of variances through a centralized database is central toward developing a robust patient safety/quality assurance program. Anonymous reporting fosters a non-punitive environment, and promotes the 'safety culture'. The goal of such a system is to review trends in clinical processes and ultimately to improve safety/quality by reducing variances associated with these processes.

Effect of amiodarone on Kv1.4 channel C-type inactivation: comparison of its effects with those induced by propafenone and verapamil.

As the major component of I(to) (slow), Kv1.4 channel plays an important role in repolarization of cardiac myocytes. C-type inactivation is one of Kv1.4 inactivation and can be affected by open channel blockers. We used the two-electrode voltage clamp technique to observe the effect of amiodarone on Kv1.4 C-type inactivation and compare amiodarone's effects on Kv1.4 with propafenone and verapamil. Our data show that those three antiarrhythmic drugs blocked fKv1.4 delta N (N-terminal deleted Kv1.4 channel from ferret heart) in voltage- and frequent-dependent manners. The amiodarone's IC50 was 489.23 +/- 4.72 microM, higher than that of propafenone (98.97 +/- 1.13 microM) and verapamil (263.26 +/- 6.89 microM) for fKv1.4 delta N channel (+50 mV). After application of amiodarone, propafenone and verapamil, fKv1.4 delta N inactivation becomes bi-exponential: the faster portion of inactivation (drug-induced inactivation) and the slower portion of inactivation (C-type inactivation). Amiodarone and verapamil fastened C-type inactivation in fKv1.4 delta N, but propafenone did not. Unlike propafenone that had no effect on fKv1.4 delta N recovery, amiodarone and verapamil slowed recovery in fKv1.4 delta N.

Effect of propafenone on Kv1.4 inactivation.

Interactions between antiarrhythmic drugs and ion channels are important subjects in the field of cardiovascular electro-pharmacology. This study explores the relationship between propafenone and C-type inactivation of Kv1.4 channel. fKvl.4deltaN, a ferret Kv1.4 N-terminal deleted mutant, was employed in this study. fKvl.4deltaN cRNA was injected into Xenopus oocytes to express fKvl.4deltaN channel and two electrode voltage clamp technique was used to record the current. We found that fKvl.4deltaN channel current was rapidly depressed in a frequency-dependent manner and meanwhile, C-type inactivation in this channel was increased more than 7 folds in the presence of 100 microM propafenone. While propafenone has no effect on Kv1.4deltaN recovery. All the results indicate that propafenone blocks Kvl.4deltaN channel through intracellular bindings and that binding of propafenone with Kvl.4deltaN channel leads to a conformational change on the extracellular site which accelerates C-type inactivation, suggesting that propafenone, as an open channel blocker, may affect the mechanism of C-type inactivation.

A novel mechanism for myocardial stunning involving impaired Ca(2+) handling.

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca(2+) responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca(2+) handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3+/-0.4%) compared with normal myocytes (8.8+/-0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131+/-8 ms) compared with normal myocytes (105+/-5 ms). The impaired contractile function was associated with decreased Ca(2+) transients (stunned, 0.33+/-0.04 versus normal, 0.49+/-0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca(2+)-current density (stunned, -4.8+/-0.4 versus normal, -6.6+/-0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca(2+) transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca(2+) handling and dephosphorylation of phospholamban, but not TnI degradation.

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes.

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.

Inactivation of voltage-gated cardiac K+ channels.

Inactivation is the process by which an open channel enters a stable nonconducting conformation after a depolarizing change in membrane potential. Inactivation is a widespread property of many different types of voltage-gated ion channels. Recent advances in the molecular biology of K+ channels have elucidated two mechanistically distinct types of inactivation, N-type and C-type. N-type inactivation involves occlusion of the intracellular mouth of the pore through binding of a short segment of residues at the extreme N-terminal. In contrast to this "tethered ball" mechanism of N-type inactivation, C-type inactivation involves movement of conserved core domain residues that result in closure of the external mouth of the pore. Although C-type inactivation can show rapid kinetics that approach those observed for N-type inactivation, it is often thought of as a slowly developing and slowly recovering process. Current models of C-type inactivation also suggest that this process involves a relatively localized change in conformation of residues near the external mouth of the permeation pathway. The rate of C-type inactivation and recovery can be strongly influenced by other factors, such as N-type inactivation, drug binding, and changes in [K+]o. These interactions make C-type inactivation an important biophysical process in determining such physiologically important properties as refractoriness and drug binding. C-type inactivation is currently viewed as arising from small-scale rearrangements at the external mouth of the pore. This review will examine the multiplicity of interactions of C-type inactivation with N-terminal-mediated inactivation and drug binding that suggest that our current view of C-type inactivation is incomplete. This review will suggest that C-type inactivation must involve larger-scale movements of transmembrane-spanning domains and that such movements contribute to the diversity of kinetic properties observed for C-type inactivation.

Voltage-dependent, open channel blockade of the cardiac sarcoplasmic reticulum potassium channel by 4-aminopyridine.

The nature of open state block was characterized in isolated canine cardiac sarcoplasmic reticulum (SR) potassium channel incorporated into planar lipid bilayers. 4-Aminopyridine (4-AP) blocked the open conductance state of the potassium channels in a voltage-dependent manner. Blockade was reversible, occurred from either the cis (cytoplasmic) or the trans (lumenal) side and was competitive with potassium ions. Reversal potential measurements indicated that this channel was impermeable to 4-AP. Measured effective electrical distances were roughly symmetrical and indicated penetration of 0.39 and 0.42 of the membrane electrical field from the cis and trans sides, respectively. Effective electrical distance was insensitive to potassium ion concentration in the range 50 to 200 mM and indicated that 4-AP was able to penetrate relatively deeply into the pore compared with blockade of sarcolemmal potassium channels. Potassium ion concentration and voltage dependence of 4-AP blockade were consistent with a two binding site blockade model, similar to the model used previously to describe calcium ion blockade of the SR potassium ion channel. Unlike calcium blockade, however, 4-AP blocked from either cis or trans in a similar manner, suggesting a distinct binding site for each of these two blockers. Open channel, voltage-dependent blockade of the SR potassium channel by 4-AP is in marked contrast to its action on sarcolemmal potassium channels and suggests that either 4-AP penetrates much farther into the potassium channel permeation pathway than was previously believed, or the SR potassium channel has a very different physical pore arrangement from that of sarcolemmal potassium channels.

Models of potassium channel inactivation: implications for open channel block.

Inactivation is a widespread property of voltage-gated ion channels. Recent molecular biological advances in the potassium channel field have elucidated two mechanistically distinct types of inactivation, N-type and C-type. Both of these mechanisms are partially coupled to activation and are usually voltage insensitive once activation is complete. This study compared the effects of a hypothetical open channel blocker on macroscopic currents by using two different models of the same cardiac transient outward current channel. Model 1 is a Hodgkin-Huxley-like model in which inactivation is an independent voltage-sensitive process. Model 2 is a model in which inactivation is voltage insensitive but is partially coupled to activation. Both models have been shown to reproduce closely the experimentally observed current. However, when modelling open channel block, the two models can differ substantially in their equilibrium degree of drug binding. This difference in equilibrium can make substantial changes in the rate of current recovery in subsequent depolarizations. It is shown that, for a rapid series of depolarizations, the time course of development of block and the degree of steady state block can differ substantially between the two models. In conclusion, molecular mechanisms of inactivation must be taken into account when modelling conformation-specific drug binding and use dependence.

Modulation of HERG affinity for E-4031 by [K+]o and C-type inactivation.

Rectification of HERG is due to a rapid inactivation process that has been labeled C-type inactivation and is believed to be due to closure of the external mouth of the pore. We examined the effects of mutation of extracellular residues that remove C-type inactivation on binding of the intracellularly acting methanesulfonanilide drug E-4031. Removal of inactivation through mutation reduced drug affinity by more than an order of magnitude. Elevation of [K+]o in the wild-type channel reduces channel affinity for E-4031. Elevation of [K+]o also interferes with the extracellular pore mouth closure associated with C-type inactivation through a 'foot in the door' mechanism. We examined the possibility that [K+]o elevation reduces drug binding through inhibition of C-type inactivation by comparing drug block in the wild-type and inactivation-removed mutant channels. Elevation of [K+]o decreased affinity in both channel constructs by a roughly equal amount. These results suggest that [K+]o alters drug binding affinity independently of its effects on C-type inactivation. They further suggest that inhibition of pore mouth closure by elevated [K+]o does not have same effect on drug affinity as mutations removing C-type inactivation.

A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes.

1. The human ether à-go-go-related gene (HERG) encodes a K+ channel that is believed to be the basis of the delayed rectified current, IKr, in cardiac muscle. We studied HERG expressed in Xenopus oocytes using a two-electrode and cut-open oocyte clamp technique with [K+]0 of 2 and 98 mM. 2. The time course of activation of the channel was measured using an envelope of tails protocol and demonstrated that activation of the heterologously expressed HERG current (IHERG) was sigmoidal in onset. At least three closed states were required to reproduce the sigmoid time course. 3. The voltage dependence of the activation process and its saturation at positive voltages suggested the existence of at least one relatively voltage-insensitive step. A three closed state activation model with a single voltage-insensitive intermediate closed state was able to reproduce the time and voltage dependence of activation, deactivation and steady-state activation. Activation was insensitive to changes in [K+]0. 4. Both inactivation and recovery time constants increased with a change of [K+]0 from 2 to 98 mM. Steady-state inactivation shifted by approximately 30 mV in the depolarized direction with a change from 2 to 98 mM K+0. 5. Simulations showed that modulation of inactivation is a minimal component of the increase of this current by [K+]0, and that a large increase in total conductance must also occur.

Hodgkin-Huxley and partially coupled inactivation models yield different voltage dependence of block.

K+ channel blockers have been shown to exhibit complex time- and voltage-dependent effects on cardiac K+ currents. Whereas much attention has been focused on the state dependence of K+ channel block, how a particular channel model can alter the predicted time and voltage dependence of channel block remains unexplored. In this study, using two different model formalisms for the same cardiac transient outward current channel, we compare the effects of a theoretical open-state specific channel blocker on macroscopic currents. Model 1 is a Hodgkin-Huxley-like model, in which inactivation is an intrinsically voltage-dependent process and occurs independently of activation. Model 2 is a "partially coupled" model, in which inactivation is intrinsically voltage insensitive but requires channel activation before it can proceed. In the absence of drug (blocking agent), the two models reproduce the macroscopic current data. In the presence of blocking agent, the two models can differ substantially, with model 1 displaying much less block than model 2. We also examine simple mathematically convenient modifications to the Hodgkin-Huxley formalism, which reproduce some, but not all, of the use-dependent properties of block. Thus model formalism is important for analysis and simulation of state-specific drug-channel interactions.

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, alpha-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 by Kv1.2, 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.

The beta subunit, Kv beta 1.2, acts as a rapid open channel blocker of NH2-terminal deleted Kv1.4 alpha-subunits.

A recently discovered class of ancillary subunits has been shown to modify the inactivation properties of alpha-subunits belonging to the Kv1 family of potassium channels. One of these subunits, Kv beta 1.2, modifies intrinsic alpha-subunit C-type inactivation. N-type inactivation and open channel block have been proposed to increase the rate of development of C-type inactivation. We demonstrate here that Kv beta 1.2 has kinetic properties which are consistent with rapid open channel block.

The N-terminal domain of a K+ channel beta subunit increases the rate of C-type inactivation from the cytoplasmic side of the channel.

Voltage-gated K+ channels are complexes of membrane-bound, ion-conducting alpha and cytoplasmic ancillary (beta) subunits. The primary physiologic effect of coexpression of alpha and beta subunits is to increase the intrinsic rate of inactivation of the alpha subunit. For one beta subunit, Kv beta 1.1, inactivation is enhanced through an N-type mechanism. A second beta subunit, Kv beta 1.2, has been shown to increase inactivation, but through a distinct mechanism. Here we show that the degree of enhancement of Kv beta 1.2 inactivation is dependent on the amino acid composition in the pore mouth of the alpha subunit and the concentration of extracellular K+. Experimental conditions that promote C-type inactivation also enhance the stimulation of inactivation by Kv beta 1.2, showing that this beta subunit directly stimulates C-type inactivation. Chimeric constructs containing just the nonconserved N-terminal region of Kv beta 1.2 fused with an alpha subunit behave in a similar fashion to coexpressed Kv beta 1.2 and alpha subunit. This shows that it is the N-terminal domain of Kv beta 1.2 that mediates the increase in C-type inactivation from the cytoplasmic side of the pore. We propose a model whereby the N terminus of Kv beta 1.2 acts as a weakly binding "ball" domain that associates with the intracellular vestibule of the alpha subunit to effect a conformational change leading to enhancement of C-type inactivation.

Time, voltage and ionic concentration dependence of rectification of h-erg expressed in Xenopus oocytes.

The rapid delayed rectifier, IKr, is believed to have h-erg (human ether-à-go-go related gene) as its molecular basis. A recent study has shown that rectification of h-erg involves a rapid inactivation process that involves rapid closure of the external mouth of the pore or C-type inactivation. We measured the instantaneous current to voltage relationship for h-erg channels using the saponin permeabilized variation of the cut-open oocyte clamp technique. In contrast to C-type inactivation in other voltage-gated K+ channels, the rate of inactivation was strongly voltage dependent at depolarized potentials. This voltage dependence could be modulated independently of activation by increasing [K+]0 from 2 to 98 mM. These results suggest that inactivation of h-erg has its own intrinsic voltage sensor.

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.

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.

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.

Time- and voltage-dependent modulation of a Kv1.4 channel by a beta-subunit (Kv beta 3) cloned from ferret ventricle.

In mammals, voltage-gated K+ channels can be made of complexes containing alpha-subunits similar to the Shaker K+ channel and smaller cytoplasmic beta-subunits. Recent studies have suggested that these ancillary beta-subunits can modulate K+ channel gating properties. We studied the effects of a K+ channel beta-subunit, Kv beta 3, coexpressed with a Kv1.4 alpha-subunit, FK1, on the time and voltage dependence of channel activation, inactivation, recovery from inactivation, and deactivation, using an oocyte expression system. Kv beta 3 was found to accelerate both the fast and the slow component of Kv1.4 inactivation. Kv beta 3 also altered the relative contributions of the two components of inactivation by increasing the contribution of the slow component to the inactivation process. Kv beta 3 slowed recovery from inactivation for Kv1.4, but not for a Kv1.4 deletion mutant lacking N-type inactivation. Finally, steady-state activation and the time course of Kv1.4 current activation were not strongly influenced by Kv beta 3; however, deactivation was slowed in the presence of Kv beta 3. This study suggests that Kv beta 3 alters channel states which follow activation.