Biomedical subjects

Glenna C L Bett

Publications and source records attributed to Glenna C L Bett.

11 recordsLinked to original sources

PubMed2006

Ancillary subunits and stimulation frequency determine the potency of chromanol 293B block of the KCNQ1 potassium channel.

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha-subunit of a voltage-gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue-specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage-dependent channel. In epithelia, KCNQ1 co-assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100-fold (IC(50) for KCNQ1 = 65.4 +/- 1.7 microm; KCNQ1/KCNE1 = 15.1 +/- 3.3 microm; KCNQ1/KCNE3 = 0.54 +/- 0.18 microm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency-dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1-like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit-mediated changes in gating can be dissociated from subunit-mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side-effects such as a predisposition to acquired long QT syndrome.

Amino Acid Sequence↗

PubMed2006

KChIP2b modulates the affinity and use-dependent block of Kv4.3 by nifedipine.

Rapidly activating Kv4 voltage-gated ion channels are found in heart, brain, and diverse other tissues including colon and uterus. Kv4.3 can co-assemble with KChIP ancillary subunits, which modify kinetic behavior. We examined the affinity and use dependence of nifedipine block on Kv4.3 and its modulation by KChIP2b. Nifedipine (150 microM) reduced peak Kv4.3 current approximately 50%, but Kv4.3/KChIP2b current only approximately 27%. Nifedipine produced a very rapid component of open channel block in both Kv4.3 and Kv4.3/KChIP2b. However, recovery from the blocked/inactivated state was strongly sensitive to KChIP2b. Kv4.3 Thalf,recovery was slowed significantly by nifedipine (120.0+/-12.4 ms vs. 213.1+/-18.2 ms), whereas KChIP2b eliminated nifedipine's effect on recovery: Kv4.3/KChIP2b Thalf,recovery was 45.3+/-7.2 ms (control) and 47.8+/-8.2 ms (nifedipine). Consequently, Kv4.3 exhibited use-dependent nifedipine block in response to a series of depolarizing pulses which was abolished by KChIP2b. KChIPs alter drug affinity and use dependence of Kv4.3.

Animals↗

PubMed2005

Time- and voltage-dependent components of Kv4.3 inactivation.

Kv4.3 inactivation is a complex multiexponential process, which can occur from both closed and open states. The fast component of inactivation is modulated by the N-terminus, but the mechanisms mediating the other components of inactivation are controversial. We studied inactivation of Kv4.3 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Inactivation during 2000 ms pulses at potentials positive to the activation threshold was described by three exponents (46 +/- 3, 152 +/- 13, and 930 +/- 50 ms at +50 mV, n = 7) whereas closed-state inactivation (at potentials below threshold) was described by two exponents (1079 +/- 119 and 3719 +/- 307 ms at -40 mV, n = 9). The fast component of open-state inactivation was dominant at potentials positive to -20 mV. Negative to -30 mV, the intermediate and slow components dominated inactivation. Inactivation properties were dependent on pulse duration. Recovery from inactivation was strongly dependent on voltage and pulse duration. We developed an 11-state Markov model of Kv4.3 gating that incorporated a direct transition from the open-inactivated state to the closed-inactivated state. Simulations with this model reproduced open- and closed-state inactivation, isochronal inactivation relationships, and reopening currents. Our data suggest that inactivation can proceed primarily from the open state and that multiple inactivation components can be identified.

Animals↗

PubMed2004

Computer model of action potential of mouse ventricular myocytes.

We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.

Action Potentials↗

PubMed2003

A model of graded calcium release and L-type Ca2+ channel inactivation in cardiac muscle.

We have developed a model of Ca(2+) handling in ferret ventricular myocytes. This model includes a novel L-type Ca(2+) channel, detailed intracellular Ca(2+) movements, and graded Ca(2+)-induced Ca(2+) release (CICR). The model successfully reproduces data from voltage-clamp experiments, including voltage- and time-dependent changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), L-type Ca(2+) channel current (I(CaL)) inactivation and recovery kinetics, and Ca(2+) sparks. The development of graded CICR is critically dependent on spatial heterogeneity and the physical arrangement of calcium channels in opposition to ryanodine-sensitive release channels. The model contains spatially distinct subsystems representing the subsarcolemmal regions where the junctional sarcoplasmic reticulum (SR) abuts the T-tubular membrane and where the L-type Ca(2+) channels and SR ryanodine receptors (RyRs) are localized. There are eight different types of subsystems in our model, with between one and eight L-type Ca(2+) channels distributed binomially. This model exhibits graded CICR and provides a quantitative description of Ca(2+) dynamics not requiring Monte-Carlo simulations. Activation of RyRs and release of Ca(2+) from the SR depend critically on Ca(2+) entry through L-type Ca(2+) channels. In turn, Ca(2+) channel inactivation is critically dependent on the release of stored intracellular Ca(2+). Inactivation of I(CaL) depends on both transmembrane voltage and local [Ca(2+)](i) near the channel, which results in distinctive inactivation properties. The molecular mechanisms underlying many I(CaL) gating properties are unclear, but [Ca(2+)](i) dynamics clearly play a fundamental role.

Animals↗

PubMed2003

Inactivation and recovery in Kv1.4 K+ channels: lipophilic interactions at the intracellular mouth of the pore.

C-type inactivation is present in many voltage-gated potassium channels and is probably related to 'slow' inactivation in calcium and sodium channels. The mechanisms underlying C-type inactivation are unclear, but it is sensitive to mutations on both the extra- and intracellular sides of the channel. We used an N-terminal deleted channel with a valine to alanine point mutation at the intracellular side of S6 (fKv1.4[V561A]DeltaN). This construct alters recovery from inactivation and inverts the relationship between C-type inactivation and [K(+)](o). We used this inverted relationship to examine C-type inactivation and coupling mechanisms between N- and C-type inactivation. The valine to alanine mutation reduces the channel's affinity for both quinidine and the N-terminal domain. However, binding of the N-terminal or quinidine restores normal recovery from inactivation. This suggests that coupling between N- and C-type inactivation is dominated by allosteric mechanisms. The permeation mechanism, driven by a reduction in permeant [K(+)](o) following pore block (which would retard C-type inactivation), contributes minimally to coupling in these channels. We propose that the cytoplasmic half of S6 forms part of the N-terminal binding site, as previously predicted from X-ray crystallography studies in the distantly related KcsA channel. Binding of the N-terminal domain or a positively charged lipophilic compound such as quinidine interacts with the hydrophobic moieties on S6 in the bound state. This binding can orientate S6 into a conformation which resembles the normal C-type inactivated state. This is the probable mechanism by which drug or N-terminal binding increases the rate of C-type inactivation via an allosteric mechanism.

Alanine↗

PubMed2003

C-type inactivation involves a significant decrease in the intracellular aqueous pore volume of Kv1.4 K+ channels expressed in Xenopus oocytes.

Channels are water-filled membrane-spanning proteins, which undergo conformational changes as they gate, i.e. open or close. These conformational changes affect both the shape of the channel and the volume of the water-filled pore. We measured the changes in pore volume associated with activation, deactivation, C-type inactivation and recovery in an N-terminal-deleted mutant of the Kv1.4 K+ channel (Kv1.4DeltaN) expressed in Xenopus oocytes. We used giant-patch and cut-open oocyte voltage clamp techniques and applied solutes which are too large to enter the pore mouth to exert osmotic pressure and thus favour smaller pore volume conformations. Applied intracellular osmotic pressure (300 mM sucrose) sped inactivation (time constants (tauinactivation): control, 0.66 +/- 0.09 s; hyperosmotic solution, 0.29 +/- 0.04 s; n = 5, P < 0.01), sped deactivation (taudeactivation: control, 18.8 +/- 0.94 ms; hyperosmotic solution, 8.01 +/- 1.92 ms; n = 5, P < 0.01), and slowed activation (tauactivation: control, 1.04 +/- 0.05 ms; hyperosmotic solution, 1.96 +/- 0.31 ms; n = 5, P < 0.01). These effects were reversible and solute independent. We estimated the pore volume change on inactivation to be about 4500 A3. Osmotic pressure had no effect when applied extracellularly. These data suggest that the intracellular side of the pore closes during C-type inactivation and the volume change is similar to that associated with activation or deactivation. This is also similar to the pore volume estimated from the crystal structure of KcsA and MthK K+ channels. Intracellular osmotic pressure also strongly inhibited re-opening currents associated with recovery from inactivation, which is consistent with a physical similarity between the C-type inactivated and resting closed state.

Algorithms↗

PubMed2003

Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect.

We studied quinidine block of Kv1.4DeltaN, a K(+) channel lacking N-type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non-additively) by interventions that slow C-type inactivation: [K(+)](o) elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ~10-fold, but left the effective electrical distance unchanged at ~0.15. Following rapid open channel block, there were time-dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time-dependent aspect of quinidine block was similar to recovery from normal C-type inactivation. Manipulations that prevented the channel from entering the C-type inactivated state (i.e. high [K(+)](o) or the K532Y mutation) prevented the development of the time-dependent quinidine-induced inactivation. The concentration dependence of the rapid block and the time-dependent quinidine-induced inactivation were similar, but the time-dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage-sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C-type inactivated state in a time- and voltage-dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C-type inactivated state through a voltage-dependent conformational change.

Allosteric Regulation↗

PubMed2003

Functionally-distinct proton-binding in HERG suggests the presence of two binding sites.

HERG (Human ether-à-go-go-related gene) potassium channels are crucial for cardiac action potential repolarization. HERG channels are also found in neuronal and tumor cells. The effect of pHo on HERG is of clinical significance because of changes in pH during myocardial ischemia, inflammation, and respiratory alkalosis. We present evidence for the presence of multiple proton binding sites in HERG. Extracellular protons bind rapidly and reversibly to affect both activation and deactivation. However, these effects occur in two distinct pHo ranges. The deactivation rate has a pKa of 6.76 +/- 0.02 compared to pKa of 5.50 +/- 0.02 for changes in current suppression, which suggests the presence of at least two proton binding sites on HERG with functionally distinct properties.

Animals↗

PubMed2002

Regulation of N- and C-type inactivation of Kv1.4 by pHo and K+: evidence for transmembrane communication.

Kv1.4 encodes a slowly recovering transient outward current (I(to)), which inactivates by a fast N-type (intracellular ball and chain) mechanism but has slow recovery due to C-type inactivation. C-type inactivation of the NH(2)-terminal deletion mutant (fKv1.4DeltaN) was inhibited by 98 mM extracellular K(+) concentration ([K(+)](o)), whereas N-type was unaffected. In 98 mM [K(+)](o), removal of intracellular K(+) concentration ([K(+)](i)) speeded C-type inactivation but had no effect on N-type inactivation, suggesting that C-type inactivation is sensitive to K(+) binding to intracellular sites. C-type inactivation is thought to involve closure of the extracellular pore mouth. However, a valine to alanine mutation on the intracellular side of S6 (V561A) of fKv1.4DeltaN alters recovery and results in anomalous speeding of C-type inactivation with increasing [K(+)](o). Extracellular pH (pH(o)) modulated both N- and C-type inactivation through an S5-H5 linker histidine (H508) with acidosis speeding both N- and C-type inactivation. Mutation of an extracellular lysine to a tyrosine (K532Y) slowed C-type inactivation and inhibited the pH dependence of both N- and C-type inactivation. These results suggest that mutations, [K(+)], and pH modulate inactivation through membrane-spanning mechanisms involving S6.

Alanine↗

PubMed2002

Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes.

The effects of the cholinergic muscarinic agonist carbachol (CCh) on the basal L-type calcium current, I(Ca,L), in ferret right ventricular (RV) myocytes were studied using whole cell patch clamp. CCh produced two major effects : (i) in all myocytes, extracellular application of CCh inhibited I(Ca,L) in a reversible concentration-dependent manner; and (ii) in many (but not all) myocytes, upon washout CCh produced a significant transient stimulation of I(Ca,L) ('rebound stimulation'). Inhibitory effects could be observed at 1 x 10(-10) M CCh. The mean steady-state inhibitory concentration-response relationship was shallow and could be described with a single Hill equation (maximum inhibition = 34.5 %, IC50 = 4 x 10(-8) M, Hill coefficient n = 0.60). Steady-state inhibition (1 or 10 microM CCh) had no significant effect on I(Ca,L) selectivity or macroscopic (i) activation characteristics, (ii) inactivation kinetics, (iii) steady-state inactivation or (iv) kinetics of recovery from inactivation. Maximal inhibition of nitric oxide synthase (NOS) activity (preincubation of myocytes in 1 mM L-NMMA (N(G)-monomethyl-L-arginine) + 1 mM L-NNA (N(G)-nitro-L-arginine) for 2-3 h plus inclusion of 1 mM L-NMMA + 1 mM L-NNA in the patch pipette solution) produced no significant attenuation of the CCh-mediated inhibition of I(Ca,L). Protocols involving (i) the nitric oxide (NO) scavenger PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; 200 microM), (ii) imposition of a 'cGMP clamp' (100 microM 8-Bromo-cGMP), and (iii) inhibition of soluble guanylyl cyclase (ODQ (1H-[1,2,4,]oxadiazolo(4,3,-a)quinoxalin-1-one), 50 microM) all failed to attenuate CCh-mediated inhibition of I(ca,L). While CCh consistently inhibited basal I(Ca,L) in all RV myocytes studied, not all myocytes displayed rebound stimulation upon CCh washout. However, there was no difference between CCh-mediated inhibition of I(Ca,L) between these two RV myocyte types, and in myocytes displaying rebound stimulation neither ODQ nor 8-Bromo-cGMP (8-Br-cGMP) altered the effect. We conclude that NO production, activation of soluble guanylyl cyclase, or changes in intracellular cGMP levels are not obligatorily involved in muscarinic-mediated modulation of basal I(Ca,L) in ferret RV myocytes.

Animals↗