Myocyte Excitability Research Articles

Polyunsaturated Fatty Acid Modulation of Voltage-Gated Ion Channels

Arachidonic acid (AA) was found to inhibit the function of whole-cell voltage-gated (VG) calcium currents nearly 16 years ago. There are now numerous examples demonstrating that AA and other polyunsaturated fatty acids (PUFAs) modulate the function of VG ion channels, primarily in neurons and muscle cells. We will review and extract some common features about the modulation by PUFAs of VG calcium, sodium, and potassium channels and discuss the impact of this modulation on the excitability of neurons and cardiac myocytes. We will describe the fatty acid nature of the membrane, how fatty acids become available to function as modulators of VG channels, and the physiologic importance of this type of modulation. We will review the evidence for molecular mechanisms and assess our current understanding of the structural basis for modulation. With guidance from research on the structure of fatty acid binding proteins, the role of lipids in gating mechanosensitive (MS) channels, and the impact of membrane lipid composition on membrane-embedded proteins, we will highlight some avenues for future investigations.

Electrophysiological properties and expression of the delayed rectifier potassium (ERG) channels in the heart of thermally acclimated rainbow trout

In ectotherms, compensatory changes in ion channel number and activity are needed to maintain proper cardiac function at variable temperatures. The rapid component of the delayed rectifier K+ current (IKr) is important for repolarization of cardiac action potential and, therefore, crucial for regulation of cellular excitability and heart rate. To examine temperature plasticity of cardiac IKr, we cloned the ether-à-go-go-related gene (ERG) channel and measured its electrophysiological properties in thermally acclimated rainbow trout (Oncorhynchus mykiss; omERG). The present findings demonstrate a complete thermal compensation in the whole cell conductance of the atrial IKr in rainbow trout acclimated to 4 degrees C (cold acclimation) and 18 degrees C (warm acclimation). In situ hybridization indicates that transcripts of the omERG channel are present throughout the muscular tissue of the heart, and quantitative PCR shows increased expression of the omERG in cold-acclimated trout compared with warm-acclimated trout. In both acclimation groups, omERG expression is higher in atrium than ventricle. In addition, the omERG has some functional features that support IKr activity at low temperatures. Voltage dependence of steady-state activation is completely resistant to temperature changes, and steady-state inactivation and activation kinetics are little affected by temperatures below 11 degrees C. Collectively, these findings suggest that high density of cardiac IKr is achieved by cold-induced increase in the number of functional omERG channels and inherent insensitivity of the omERG to temperature below 11 degrees C. These adaptations are probably important in maintaining high heart rates and proper excitability and contractility of trout cardiac myocytes in the cold.

Analysis of Dihydropyridine and Ryanodine Receptor Clusters in Healthy and Failing Human Cardiac Myocytes

Excitation contraction coupling in cardiac ventricular muscle is regulated through calcium-induced calcium release (CICR). This process is where the action potential triggers the opening of voltage sensitive calcium channel or dyhyropyridine receptor (DHPR) on the plasma or t-tubular membrane of cardiac myocytes. This allows a small influx of Ca2+ (L-type current) into the cardiac cells which then induces the opening of the sarcoplasmic reticulum ryanodine receptor causing a much larger release of Ca2+ in the form of a calcium spark. Propagation of these sparks throughout the cell allows cytoplasmic build up of Ca2+ that in turn triggers muscle contraction. The close association of DHPR and RYR2 in a structure termed the dyad is critical to successful CICR. Several animal models of heart failure have demonstrated dyssynchronous Ca2+ release is associated with disrupted t-tubular network including a decrease in DHPR/RYR colocalisation. We hypothesised that similar disorganisation in myocyte excitation–contraction-coupling mechanisms in insulinresistant cardiomyopathy. Cardiac specific GLUT4 knock-out (GLUT4-KO)mice and their littermate controls which show a global GLUT4 knock-down (GLUT4-KD) were used for this study. We have previously reported that GLUT4-KO and GLUT4KD exhibit >95% and 85% reduction in GLUT4 expression respectively, relative to wild-type C57Bl6. Cardiomyocytes were isolated enzymatically from hearts of 15 to 35-week-old male mice. The GLUT4-KO mice exhibited significantly greater heart weight (380.5± 16.8 vs. 217± 6.0mg, p< 0.001) and cardiacweight index (14.2± 0.6 vs. 7.8± 0.2mg/g, p< 0.001) compared toGLUT4-KDmice. Myocyte Ca2+ currents and Na+/Ca2+ exchanger currents were measured using whole cell patch clamp recording techniques, and normalized for myocyte size determined by capacitance measurement (pA/pF). Mean capacitance of GLUT4-KO myocytes was significantly greater than GLUT4-KD(437± 28vs. 269± 15pF,p< 0.0001). InGLUT4KO, peak voltage-activated (holding potential −90mV) Ca2+ channel current density was significantly reduced compared with GLUT4-KD (−2.82± 0.3 vs. −5.33± 0.7 pA/pF, p< 0.05). The Ni-sensitive Na+/Ca2+ exchange currents were significantly larger in the GLUT4-KO in both inward (−1.56± 0.29 vs. −0.83± 0.16 pA/pF, p< 0.05) and outward modes (1.82± 0.25 vs. 1.09± 0.1 pA/pF, p< 0.02). Thus, cardiac-specific knockout of the GLUT4 trans-

Increased cardiomyocyte function and Ca 2+ transients in mice during early congestive heart failure

End-stage heart failure is believed to involve depressed cardiomyocyte contractility and Ca 2+ transients. However, the time course of these alterations is poorly understood. We examined alterations in myocyte excitation–contraction coupling in a mouse model of early congestive heart failure (CHF) following myocardial infarction. One week after myocardial infarction was induced by ligation of the left coronary artery, CHF mice were selected based on established criteria (increased left atrial diameter, increased lung weight). Sham-operated animals (SHAM) served as controls. Echocardiographic measurements showed decreased global function in early CHF relative to SHAM, but increased local function in viable regions of the myocardium which deteriorated with time. Cardiomyocytes isolated from the non-infarcted septum also exhibited larger contractions in early CHF than SHAM (CHF = 219.6 ± 15.3% of SHAM values, P < 0.05; 1 Hz field stimulation), and relaxation was more rapid (time to 50% relaxation = 82.9 ± 5.5% of SHAM values, P < 0.05). Ca 2+ transients (fluo-4 AM) were larger and decayed more rapidly in CHF than SHAM during both field stimulation (1 Hz) and voltage-clamp steps. Sarcoplasmic reticulum (SR) Ca 2+ content was increased. Western blots showed that while SR Ca 2+ ATPase (SERCA) expression was unaltered in CHF, phospholamban (PLB) was downregulated (60 ± 11% of SHAM values, P < 0.05). Thus, an increased SERCA/PLB ratio in CHF may promote SR Ca 2+ re-uptake. Additionally, peak L-type Ca 2+ current and Na +/Ca 2+ exchanger expression were increased in CHF, suggesting increased sarcolemmal Ca 2+ flux. Thus, in early CHF, alterations in Ca 2+ homeostasis improve cardiomyocyte contractility which may compensate for loss of function in the infarction area.

Diabetes‐related alterations in cAMP‐mediated potassium channel currents in short term cultures of detrusor myocytes

The goal of these studies was to further explore the myogenic basis for diabetes (DM)-related bladder dysfunction. To this end, we evaluated cAMP-mediated K+ channel function and regulation in short term cultures of detrusor myocytes derived from urothelial denuded bladders of 2-month streptozotocin (STZ)-diabetic (DM) and age-matched control rats. Cell cultures (passages 1–3) were derived from male F344 rats in the following 3 groups, DM (n= 4 rats), insulin (IN)-treated DM (n=4 rats), and an age-matched control group (AMC; n=4 rats). Cells were voltage clamped at −70 mV and I–V curves were performed in 10 mV increments ranging from −60 mV to + 100 mV. Cells were subjected to 6 consecutive I–V curves. Following a control I–V curve, cells were exposed to the following drugs at 10–15 minute intervals: 1 mM 8-Br-cAMP, 100 nM IBTX (BK block), 10 μM glybenclamide (KATP block), 100 μM 4-AP (Kv block), and 100 nM apamin (SK block). An IN-reversible increase in resting membrane potential, and an IN-reversible decrease in cAMP-induced K+ currents and current density were observed (p<0.05, 2-Way ANOVA). A significant DM-related decline in the IBTX-sensitive portion of the whole cell K+ current, and a significant increase in the 4-AP sensitive-portion (p<0.05) was noted. These studies document significant DM-related alterations in K+ currents that are consistent with increased detrusor myocyte excitability (DK60037).

A new multi-scale simulation model of the circulation: from cells to system

We developed a comprehensive cell model that simulates the sequential cellular events from membrane excitation to contraction in the human ventricle. By combining this ventricular cell model with a lumped circulation model, we examined how blood pressure dynamics in the ventricle and aorta are related to the cellular processes. To convert cell contraction into ventricular pressure using Laplace's law, we introduced a simple geometric model of a ventricle: one shaped like a thin-walled hemisphere. The force of contraction of a single cell induces tension in the hemispheric ventricular wall, which generates the ventricular and aortic pressures in the lumped circulation model. The time courses of the hemodynamic properties, as well as the volume-pressure trajectory of the left ventricle, were well reproduced. Our multi-scale cardiovascular model, which covers from cardiac cells to the circulatory system, simulates the typical characteristics of heart mechanics, such as the pressure-volume relationship, stroke volume and the effect of the increased maximum free calcium concentration on cardiovascular hemodynamics. To test the cell-circulation coupling characteristics of the model, we simulated the effects of a decrease in L-type calcium channel conductance (cell level) on left ventricular pressure (system level). The variation due to different pacing frequencies for myocyte excitation was also investigated to assess the effects of heart rate on cardiac cells and the circulatory system.

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na(v)1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na(v)1.2, Na(v)1.3, and Na(v)1.5 indicated that mouse brain Nedd4-2 binds to the Na(v) PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na(v)1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K(d) of approximately 55 muM. We tested the binding properties and the ability to ubiquitinate and downregulate Na(v)1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na(v)1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na(v)1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na(v)1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

Fish intake and risk of incident atrial fibrillation

Study Question: Does fish intake influence the incident rate of atrial fibrillation? Methods: The Cardiovascular Health Study, a population-based prospective cohort study, was used to prospectively identify subjects with new-onset atrial fibrillation (AF) on the basis of hospital discharge records and annual electrocardiograms. The fish consumption and usual dietary intake was assessed at baseline in 4815 adults ≥age 65 years in 1989 and 1990, with a mean follow-up of 12 years. Results: Mean age was 72 years, 95% were white, about 20% had diabetes and 20% had coronary disease; mean sBP was 135 mm Hg and 45% were treated for hypertension. Incident AF was detected in 980 subjects. In multivariate analyses, consumption of tuna or other broiled or baked fish was inversely associated with incidence of AF, with a 28% lower risk with intake 1 to 4 times per week (HR=0.72; 95% CI=0.58–0.91; p=0.005), and 31% lower risk with intake ≥5 times per week (HR=0.69; 95% CI=0.52–0.91; p=0.008), compared with <1 time per month (p trend=0.004). Results were not materially different after adjustment for preceding myocardial infarction (MI) or congestive heart failure. In similar analyses, fried fish/fish sandwich consumption was not associated with lower risk of AF. Conclusions: Among elderly adults, consumption of tuna or other broiled or baked fish, but not fried fish or fish sandwiches, is associated with lower incidence of AF. Fish intake may influence risk of this common cardiac arrhythmia. Perspective: The study results are consistent with the clinical studies demonstrating less sudden death in coronary disease and the putative anti-arrhythmic effects of n-3 fatty acids including decreased ventricular myocyte excitability, and termination of induced asynchronous contraction in atrial myocytes. Fish oil intake can result in replacement of cardiac cellular membrane arachidonic acid by eicosapentaenoic acid. Why not fried fish? It is not the beer because alcohol intake was not greater in the fried fish/fish sandwich group. Frying can change the n-3 to n-6 PUFAs and lead to formation of trans-fatty acids. MR

Properties of average-conductance cationic channels that mediate cholinergic excitation of guinea-pig ileum myocytes under conditions close to the physiological norm

Properties of average-conductance cationic channels that mediate cholinergic excitation of guinea-pig ileum myocytes under conditions close to the physiological norm

Simulation of ATP metabolism in cardiac excitation–contraction coupling

To obtain insights into the mechanisms underlying the membrane excitation and contraction of cardiac myocytes, we developed a computer model of excitation–contraction coupling (Kyoto model: Jpn. J. Physiol. 53 (2003) 105). This model was further expanded by incorporating pivotal reactions of ATP metabolism; the model of mitochondrial oxidative phosphorylation by Korzeniewski and Zoladz (Biophys. Chem. 92 (2001) 17). The ATP-dependence of contraction, and creatine kinase and adenylate kinase were also incorporated. After minor modifications, the steady-state condition was well established for all the variables, including the membrane potential, contraction, and the ion and metabolite concentrations in sarcoplasmic reticulum, mitochondria and cytoplasm. Concentrations of major metabolites were close to the experimental data. Responses of the new model to anoxia were similar to experimental results of the P-31 NMR study in whole heart. This model serves as a prototype for developing a more comprehensive model of excitation–contraction–metabolism coupling.

Properties of average-conductance cationic channels that mediate cholinergic excitation of guinea-pig ileum myocytes under conditions close to the physiological norm

The properties of cationic channels of an average unitary conductance were studied in guinea-pig ileum smooth muscle cells using a patch-clamp technique in the outside-out configuration. Cationic channels were activated by addition of 200 µM GTPγS to an intrapipette solution, which resulted in stable activation of G proteins. The replacement of external cesium-containing (in the absence of Ca2+ and Mg2+ ions) solution with a “more physiological” sodium solution containing 2.5 mM Ca2+ and 2.0 mM Mg2+ led to smaller values of both the amplitude of currents through the unitary cationic channels under study and the probability of the stay of the channel in the open state (Po). The drop in current amplitude was related mostly to the blocking effect of bivalent cations, while a decrease in the Po resulted from the replacement of Cs+ with Na+. Just a drop in the Po, which was responsible for approximately 85% of the inhibitory effect, played a crucial role in the suppression of the integral transmembrane current.

BDNF and CNTF regulate cholinergic properties of sympathetic neurons through independent mechanisms

Cultured neonatal sympathetic neurons can synthesize and corelease norepinephrine (NE) and acetylcholine (ACh). Evoked release of NE has an excitatory effect on the beat rate of cocultured cardiac myocytes while ACh release results in myocyte inhibition. Here we show that the cholinergic properties of the neurons and the relative level of NE and ACh corelease are modulated by neurotrophic factors. Brain-derived neurotrophic factor (BDNF) rapidly promoted ACh release in the absence of cholinergic differentiation activity and even in neurons that were predominantly noradrenergic. This increase in the cholinergic component of sympathetic cotransmission was sufficient for myocytes to display an overall inhibitory response to neuronal stimulation. In contrast, short-term growth in ciliary neurotrophic factor (CNTF) resulted in the upregulation of cholinergic and downregulation of noradrenergic markers without an effect on normal excitatory neurotransmission. Only once the cells had acquired a cholinergic phenotype did CNTF acutely promote the evoked release of the cholinergic vesicle pool. The results of this study indicate that BDNF and CNTF, acting through independent pathways, modulate NE and ACh cotransmission to regulate the level of sympathetic excitation or inhibition of cardiac myocytes.

Modulation of triggered activity by uncoupling in the ischemic border. A model study with phase 1b-like conditions.

Triggered beats during regional ischemia may depend upon the electrical source and sink charge interactions between adjacent regions of normal and ischemic cardiac tissue that are partly controlled by electrical coupling. To study these relationships, we modified parameters in the Luo-Rudy dynamic membrane equations to reflect physiologic conditions associated with phase 1b arrhythmias. Superthreshold delayed afterdepolarizations (DADs) formed after pacing. Coupling contributions were then examined using: (i) a single phase 1b myocyte connected via a variable resistance to a single normal myocyte, and (ii) a multicellular fiber with a 1-cm segment of phase 1b myocytes connected to a 1-cm normal segment having resistance changes that were confined to the ischemic segment. Integration of ionic, capacitive and coupling currents during DAD initiation allowed charge quantification. In cell pairs, phase 1b myocyte DADs were suppressed at resistances where normal myocyte pacing resulted in phase 1b myocyte excitation. Coupling charge requirements limited capacitive charging in the phase 1b myocyte, which occurred in combination with diastolic hyperpolarization that shifted transmembrane potential from threshold. In multicellular fiber simulations, DADs were suppressed with strong coupling in the phase 1b segment. Moderate uncoupling of that segment allowed superthreshold DAD formation away from the border that initiated action potential propagation in the normal segment. With severe uncoupling, propagation failed at the border. These findings support the clinical and experimental observation that intermediate uncoupling is an important contributor to phase 1b arrhythmogenesis.

The Kv2.2 alpha subunit contributes to delayed rectifier K(+) currents in myocytes from rabbit corpus cavernosum.

K(+) currents are known to regulate the excitability of corpus cavernosum myocytes and therefore to play a role in the control of penile erection and detumescence. We used electrophysiology and molecular cloning techniques to identify ion channel proteins that contribute to K(+) currents in rabbit cavernosal myocytes. Currents were recorded from freshly isolated myocytes using whole-cell patch clamp techniques. Cavernosal myocytes expressed a delayed rectifier voltage-gated K(+) current that appeared to contribute to the resting membrane potential. This voltage-gated K(+) (K(v)) current was inhibited by the nonselective compounds 4-aminopyridine (1-10 mM), (+)-fenfluramine (10 micro M-1 mM), and Grammostola spatulata venom (1:100) in a dose-dependent and reversible fashion. Hanatoxin-1 (1 micro M), a selective Kv2 channel inhibitor, partially inhibited the current, but alpha-dendrotoxin (200 nM), a Kv1 channel blocker, had no effect. The nucleotide sequence of K(+) channel subunits was determined by polymerase chain reaction-based cloning techniques using RNA derived from cavernosal muscle strips and single identified myocytes. Molecular cloning techniques identified the full-length sequence of the rabbit ortholog of the Kv2.2 alpha subunit. This sequence contains 911 amino acid residues and is 92% identical to the recently revised human Kv2.2 sequence. Identified cavernosal myocytes of the type used in physiological recordings expressed Kv2.2 messenger RNA. We conclude that Kv2.2 alpha subunits contribute to whole-cell currents in rabbit canvernosal myocytes. Further, K(v) currents play a role in regulating membrane potential and hence excitability in rabbit cavernosal myocytes.

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.

A calcium sensor in the sodium channel modulates cardiac excitability.

Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.

POTASSIUM OUTWARD CURRENTS IN FRESHLY DISSOCIATED RABBIT CORPUS CAVERNOSUM MYOCYTES

Cavernous smooth muscle cells have a key role in the control of penile erection and detumescence. In this study the types of smooth muscle cells and currents present in isolated rabbit corpus cavernosum myocytes were characterized. Immunohistochemical methods were used to identify cavernous smooth muscle cells. Currents were recorded from freshly dissociated myocytes using the whole cell and amphotericin perforated patch clamp techniques. Cavernous myocytes were identified by alpha-smooth muscle actin and smooth muscle myosin immunoreactivity. Based on electrical properties at least 2 types of myocytes were present. Type I cells showed more depolarized membrane potentials, lower capacitance, higher input resistance and increased current densities at positive potentials than type II cells. In types I and II cells at voltages positive to 30 mV, maxi K+ channel (Ca2+ activated large conductance K+ channel or BK) blockade with iberiotoxin or charybdotoxin reduced outward currents by approximately 40% to 80% at 80 mV. Maxi K+ channel blocking did not affect cell membrane potential. Type II cells showed delayed rectifier K+ channel-type outward currents that were not detected in type I cells. Delayed rectifier K+ channel-type currents were resistant to iberiotoxin or charybdotoxin, activated at approximately -50 to -40 mV. and inactivated weakly. The data suggest that cavernous smooth muscle cells are heterogeneous with at least 2 subtypes identified based on membrane potential, capacitance, input resistance, current density and delayed rectifier K+ channel expression. The activation threshold suggests that delayed rectifier K+ channels are open at the resting membrane potential and, therefore, contribute to control and regulation of the cavernous myocyte excitability.

INTEGRATIVE ERECTILE BIOLOGY: The Effects of Age and Disease on Gap Junctions and Ion Channels and Their Potential Value to the Treatment of Erectile Dysfunction

Initiation, maintenance, and modulation of corporal smooth muscle tone are critically dependent upon agonist-induced changes in intracellular calcium levels and mobilization as well as transmembrane calcium flux. The transient control of myocyte excitability and contractility at the cellular level is inextricably linked to membrane potential, which, in turn, is modulated by potassium ion efflux through one of the four known corporeal smooth muscle potassium ion channels. Corporal tissue responses are subsequently coordinated by means of the movement of intracellular second messenger molecules (i.e., IP3, cAMP, cGMP) and ions (i.e., K+ and Ca2+) among the corporal myocytes by means of intercellular communication through gap junction channels. Knowledge of the critical contribution of these interlinking cellular (nonjunctional ion channels [e.g., maxi-K]) and tissue (gap junction channels [e.g., connexin 43]) systems to the modulation of erectile capacity has provided the scientific rationale for the promulgation of the successful preclinical testing of hSlo ion channel gene therapy for the normalization of erectile status in both aged and diabetic rats.

Modulation of A-type potassium channels by a family of calcium sensors.

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Effects of troglitazone and pioglitazone on the action potentials and membrane currents of rabbit ventricular myocytes

The effects of the antidiabetic thiazolidinediones troglitazone and pioglitazone on action potentials and membrane currents were studied in rabbit ventricular myocytes. Troglitazone (10 μM) reversibly reduced excitability of the myocytes and modified their action potential configuration. It significantly increased the stimulation threshold required to elicit action potentials and decreased action potential amplitude and the maximum upstroke velocity of the action potentials. The Inhibition of the maximum upstroke velocity by troglitazone was also significant at 1 μM. Voltage-clamp experiments revealed that troglitazone (10 μM) reversibly inhibited both the slow inward Ca 2+ current and the steady-state K + current. In contrast to troglitazone, pioglitazone (1–10 μM) had no significant effect on the excitability, action potential configuration, or membrane currents of myocytes. These results suggest that troglitazone, but not pioglitazone, modulates Na +, Ca 2+ and K + currents, leading to the changes in excitability and action potential configuration of ventricular myocytes.