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Wayne R Giles

Publications and source records attributed to Wayne R Giles.

27 records · Page 2Linked to original sources
PubMed2003

A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells.

We have investigated the physiological role of the "rapidly activating" delayed rectifier K+ current (IKr) in pacemaker activity in isolated sinoatrial node (SAN) myocytes and the expression of mouse ether-a-go-go (mERG) genes in the adult mouse SAN. In isolated, voltage-clamped SAN cells, outward currents evoked by depolarizing steps (greater than -40 mV) were strongly inhibited by the class III methanesulfonanilide compound E-4031 (1-2.5 microM), and the deactivation "tail" currents that occurred during repolarization to a membrane potential of -45 mV were completely blocked. E-4031-sensitive currents (IKr) reached a maximum at a membrane potential of -10 mV and showed pronounced inward rectification at more-positive membrane potentials. Activation of IKr occurred at -40 to 0 mV, with half-activation at about -24 mV. The contribution of IKr to action potential repolarization and diastolic depolarization was estimated by determining the E-4031-sensitive current evoked during voltage clamp with a simulated mouse SAN action potential. IKr reached its peak value (approximately 0.6 pA/pF) near -25 mV, close to the midpoint of the repolarization phase of the simulated action potential, and deactivated almost completely during the diastolic interval. E-4031 (1 microM) slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% (n = 5). Expression of mRNA corresponding to three isoforms coded by the mouse ERG1 gene (mERG1), mERG1a, mERG1a', and mERG1b, was consistently found in the SAN. Our data provide the first detailed characterization of IKr in adult mouse SAN cells, demonstrate that this current plays an important role in pacemaker activity, and indicate that multiple isoforms of mERG1 can contribute to native SAN IKr.

Animals↗
PubMed2003

Two arginines in the cytoplasmic C-terminal domain are essential for voltage-dependent regulation of A-type K+ current in the Kv4 channel subfamily.

Contributions of the C-terminal domain of Kv4.3 to the voltage-dependent gating of A-type K+ current (IA) were examined by (i) making mutations in this region, (ii) heterologous expression in HEK293 cells, and (iii) detailed voltage clamp analyses. Progressive deletions of the C terminus of rat Kv4.3M (to amino acid 429 from the N terminus) did not markedly change the inactivation time course of IA but shifted the voltage dependence of steady state inactivation in the negative direction to a maximum of -17 mV. Further deletions (to amino acid 420) shifted this parameter in the positive direction, suggesting a critical role for the domain 429-420 in the voltage-dependent regulation of IA. There are four positively charged amino acids in this domain: Lys423, Lys424, Arg426, and Arg429. The replacement of the two arginines with alanines (R2A) resulted in -23 and -13 mV shifts of inactivation and activation, respectively. Additional replacement of the two lysines with alanines did not result in further shifts. Single replacements of R426A or R429A induced -15 and -10 mV shifts of inactivation, respectively. R2A did not significantly change the inactivation rate but did markedly change the voltage dependence of recovery from inactivation. These two arginines are conserved in Kv4 subfamily, and alanine replacement of Arg429 and Arg432 in Kv4.2 gave essentially the same results. These effects of R2A were not modulated by co-expression of the K+ channel beta subunit, KChIPs. In conclusion, the two arginines in the cytosolic C-terminal domain of alpha-subunits of Kv4 subfamily strongly regulate the voltage dependence of channel activation, inactivation, and recovery.

Alanine↗
PubMed2003

Comparison of time- and voltage-dependent K+ currents in myocytes from left and right atria of adult mice.

Consistent differences in K+ currents in left and right atria of adult mouse hearts have been identified by the application of current- and voltage-clamp protocols to isolated single myocytes. Left atrial myocytes had a significantly (P < 0.05) larger peak outward K+ current density than myocytes from the right atrium. Detailed analysis revealed that this difference was due to the rapidly activating sustained K+ current, which is inhibited by 100 muM 4-aminopyridine (4-AP); this current was almost three times larger in the left atrium than in the right atrium. Accordingly, 100 muM 4-AP caused a significantly (P < 0.05) larger increase in action potential duration in left than in right atrial myocytes. Inward rectifier K+ current density was also significantly (P < 0.05) larger in left atrial myocytes. There was no difference in the voltage-dependent L-type Ca2+ current between left and right atria. As expected from this voltage-clamp data, the duration of action potentials recorded from single myocytes was significantly (P < 0.05) shorter in myocytes from left atria, and left atrial tissue was found to have a significantly (P < 0.05) shorter effective refractory period than right atrial tissue. These results reveal similarities between mice and other mammalian species where the left atrium repolarizes more quickly than the right, and provide new insight into cellular electrophysiological mechanisms responsible for this difference. These findings, and previous results, suggest that the atria of adult mice may be a suitable model for detailed studies of atrial electrophysiology and pharmacology under control conditions and in the context of induced atrial rhythm disturbances.

Action Potentials↗
PubMed2003

Resting membrane potential regulates Na(+)-Ca2+ exchange-mediated Ca2+ overload during hypoxia-reoxygenation in rat ventricular myocytes.

In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na(+)-Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na(+)-Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia-reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mM yielded the following resting membrane potentials: -27.6+/-1.63 mV, -102.2+/-1.89, -86.5+/-1.03, -80.1+/-1.25, -73.6+/-1.02 and -66.4+/-1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia-reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura-2. These results demonstrated that after chemically induced hypoxia-reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mM [K+]o significantly reduced [Ca2+]i (7.5+/-0.32 vs. 16.9+/-0.55%); while depolarization (with either 0.5 or 15 mM [K+]o) significantly increased [Ca2+]i (31.8+/-3.21 and 20.8+/-0.36 vs. 16.9+/-0.55%, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na(+)-Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na(+)-Ca2+ exchanger inhibitor KB-R7943. During reoxygenation KB-R7943 (5 microM) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mM [K+]o: 2.2+/-0.40 vs. 10.8+/-0.14%) and in depolarized myocytes (in 15 mM [K+]o: -2.1+/-0.51 vs. 11.3+/-0.05%). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia-reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na(+)-Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.

Animals↗
PubMed2003

A mathematical model of the electrophysiological alterations in rat ventricular myocytes in type-I diabetes.

Our mathematical model of the rat ventricular myocyte (Pandit et al., 2001) was utilized to explore the ionic mechanism(s) that underlie the altered electrophysiological characteristics associated with the short-term model of streptozotocin-induced, type-I diabetes. The simulations show that the observed reductions in the Ca(2+)-independent transient outward K(+) current (I(t)) and the steady-state outward K(+) current (I(ss)), along with slowed inactivation of the L-type Ca(2+) current (I(CaL)), can result in the prolongation of the action potential duration, a well-known experimental finding. In addition, the model demonstrates that the slowed reactivation kinetics of I(t) in diabetic myocytes can account for the more pronounced rate-dependent action potential duration prolongation in diabetes, and that a decrease in the electrogenic Na(+)-K(+) pump current (I(NaK)) results in a small depolarization in the resting membrane potential (V(rest)). This depolarization reduces the availability of the Na(+) channels (I(Na)), thereby resulting in a slower upstroke (dV/dt(max)) of the diabetic action potential. Additional simulations suggest that a reduction in the magnitude of I(CaL), in combination with impaired sarcoplasmic reticulum uptake can lead to a decreased sarcoplasmic reticulum Ca(2+) load. These factors contribute to characteristic abnormal [Ca(2+)](i) homeostasis (reduced peak systolic value and rate of decay) in myocytes from diabetic animals. In combination, these simulation results provide novel information and integrative insights concerning plausible ionic mechanisms for the observed changes in cardiac repolarization and excitation-contraction coupling in rat ventricular myocytes in the setting of streptozotocin-induced, type-I diabetes.

Action Potentials↗
PubMed2003

Acyl coenzyme A esters differentially activate cardiac and beta-cell adenosine triphosphate-sensitive potassium channels in a side-chain length-specific manner.

Recent evidence demonstrates that long-chain acyl coenzyme A esters (CoAs) activate cardiac and beta-cell plasma-membrane (pmK(ATP)) adenosine triphosphate (ATP)-sensitive potassium channels. In this study, we have investigated the differential effects of acyl CoAs of short and medium side-chain length on cardiac and beta-cell pmK(ATP) isoforms. At the single-channel level, the addition of acyl CoAs of differing side-chain length (2 to 16 carbons) to the inside face of membrane patches from ventricular myocytes caused varying increases in pmK(ATP) channel open probability proportional to increases in acyl side-chain length (20 mumol/L acetyl CoA: 310% +/- 90%, 20 mumol/L decanoyl CoA: 570% +/- 150%). A similar dependence of activation on side-chain length was observed in recombinant pmK(ATP) channels (SUR2A/Kir6.2) with full activation of current requiring both the acyl and CoA moieties in the esterified form. We found the recombinant beta-cell K(ATP) channel (SUR1/Kir6.2) to be much less sensitive to medium-chain acyl CoAs (decanoyl CoA: 124% +/- 15% v 231% +/- 25% in SUR2A/Kir6.2), suggesting a role for the cardiac sulfonylurea receptor, SUR2A, in the molecular mechanism of activation by these compounds. We propose that fatty acid metabolism, and the resultant generation of acyl CoAs of varying side-chain length, may be an important regulator of cellular excitability via interactions with the K(ATP) channel.

ATP-Binding Cassette Transporters↗
PubMed2003

Electrophysiological evidence for a gradient of G protein-gated K+ current in adult mouse atria.

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine-sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 microM) caused a substantial increase in K+ current in all myocytes examined. The current-voltage (I-V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05). The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 microM). ACh (10 microM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 microM). Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes. These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.

Action Potentials↗
PubMed2002

Mathematical model of the rapidly activating delayed rectifier potassium current I(Kr) in rabbit sinoatrial node.

INTRODUCTION: A rapidly activating delayed rectifier potassium current (I(Kr)) is known to have an important role in determining the properties of spontaneous pacing in enzymatically isolated rabbit sinoatrial node (SAN) cells. The functional characteristics of I(Kr) are conferred by its dependence on time, voltage, and external potassium. The aim of this study was to develop a rigorous mathematical representation for I(Kr) based on experimental findings and to investigate the role of I(Kr) in the automaticity and intercellular communication of SAN cells. METHODS AND RESULTS: A Markov model was developed using available experimental data for I(Kr) in rabbit SAN. The dependence of I(Kr) on external potassium, [K+]o, was incorporated using data from both in vitro preparations and results from heterologous expression experiments for this ether-a-go-go related gene product. Our simulation results show the following. (1) I(Kr) is the dominant repolarizing current in rabbit SAN cells. (2) Deactivation of I(Kr) contributes to the net current change during the early diastolic depolarization phase. (3) Inward rectification of I(Kr) results in a decrease in membrane resistance during repolarization relative to plateau. (4) The complex [K+]o dependence of I(Kr) confers [K+]o insensitivity on isolated cells, which may account for the sensitivity of pacing rate to elevated [K+]o at the tissue level. CONCLUSION: Model results show that I(Kr) mediates diastolic depolarization by the kinetics of its decay and by lowering resistance during late repolarization. In elevated [K+]o, increased chord conductance is balanced by the changes in kinetics and voltage dependence of I(Kr) so that the pacing rate of single cells may be more [K+]o insensitive than expected. In addition, elevated [K+]o increases I(Kr) magnitude during repolarization but lowers resistance, so current flow through gap junctions is less able to hyperpolarize pacing cells.

Action Potentials↗