Ian Findlay

Inwardly Rectifying Potassium Channels: Their Structure, Function, and Physiological Roles

Inwardly rectifying K(+) (Kir) channels allow K(+) to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K(+) channels (Kir6.x) are tightly linked to cellular metabolism, and K(+) transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg(2+) and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH(2) and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.

Physiological modulation of inactivation in L‐type Ca2+ channels: one switch

The relative contributions of voltage‐ and Ca2+‐dependent mechanisms of inactivation to the decay of L‐type Ca2+ channel currents (ICaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage‐dependent inactivation (VDI) was thought to be slow and Ca2+‐dependent inactivation (CDI) resulting from Ca2+ influx and Ca2+‐induced Ca2+‐release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca2+ entry and the contribution of ICaL to the cardiac action potential. Physiological modulation of ICaL by β‐adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca2+ channel activity, Ca2+ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of ICaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β‐adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of ICaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of ICaL to the action potential will be determined largely by voltage and by Ca2+ following β‐adrenergic stimulation.

Extracellular links in Kir subunits control the unitary conductance of SUR/Kir6.0 ion channels

Potassium (K+) channels are highly selective for K+ ions but their unitary conductances are quite divergent. Although Kir6.1 and Kir6.2 are highly homologous and both form functional K+ channels with sulfonylurea receptors, their unitary conductances measured with 150 mM extracellular K+ are ∼35 and 80 pS, respectively. We found that a chain of three amino acid residues N123–V124–R125 of Kir6.1 and S113–I114–H115 of Kir6.2 in the M1–H5 extracellular link and single residues M148 of Kir6.1 and V138 of Kir6.2 in the H5–M2 link accounted for the difference. By using a 3D structure model of Kir6.2, we were able to recognize two independent plausible mechanisms involved in the determination of single channel conductance of the Kir6.0 subunits: (i) steric effects at Kir6.2V138 or Kir6.1M148 in the H5–M2 link influence directly the diffusion of K+ ions; and (ii) structural constraints between Kir6.2S113 or Kir6.1N123 in the M1–H5 link and Kir6.2R136 or Kir6.1R146 near the H5 region control the conformation of the permeation pathway. These mechanisms represent a novel and possibly general aspect of the control of ion channel permeability.

β-Adrenergic stimulation modulates Ca2+- and voltage-dependent inactivation of L-type Ca2+ channel currents in guinea-pig ventricular myocytes

The objective of this study was to examine the effect of β‐adrenergic stimulation upon voltage‐ and Ca2+‐induced inactivation of native cardiac L‐type Ca2+ channels. Whole‐cell currents were recorded from guinea‐pig isolated ventricular myocytes. Total and voltage‐dependent inactivation was separated by replacing extracellular Ca2+ with Mg2+. L‐type Ca2+ channel behaviour was monitored with outward Ca2+ channel currents. First, the voltage dependence of inactivation was studied at fixed times (50 and 1000 ms) after activation. This showed that under control conditions Ca2+ contributed little to inactivation. In isoproterenol (isoprenaline), voltage‐dependent inactivation was markedly reduced and Ca2+ contributed largely to total inactivation. Second, the time dependence of inactivation was studied at a fixed voltage (+10 mV). In control conditions the fast phase of inactivation (τf≈15 ms) was reduced to the same extent by ryanodine (τf≈30 ms) and the absence of Ca2+ (τf≈30 ms) while the slow phase of inactivation (τs≈70 ms) was reduced by ryanodine (τs≈160 ms) and further reduced in the absence of Ca2+ (τs≈300 ms). In isoproterenol, biphasic inactivation of Ca2+ currents (τf≈4 ms, τs≈60 ms) was replaced by a single slow (τ≈450 ms) phase of inactivation in the absence of Ca2+. It is concluded that, under control conditions Ca2+ channel current decay is largely dominated by rapid voltage‐dependent inactivation, while in isoproterenol this is replaced by Ca2+‐induced inactivation.

Voltage- and cation-dependent inactivation of L-type Ca2+ channel currents in guinea-pig ventricular myocytes

L‐type Ca2+ channel currents in native ventricular myocytes inactivate according to voltage‐ and Ca2+‐dependent processes. This study sought to examine the effect of β‐adrenergic stimulation on the contributions of voltage and Ca2+ to Ca2+ current decay. Ventricular myocytes were enzymatically isolated from guinea‐pig hearts. Inward whole‐cell Cd2+‐sensitive L‐type Ca2+ channel currents were recorded with the patch clamp technique and comparison was made between inward currents carried by Ca2+ and either Ba2+, Sr2+ or Na+. In control conditions the decay of Ca2+ currents was faster than Ba2+, Sr2+ or Na+ currents at negative voltages while at positive voltages there was no difference. The relationship between voltage and inactivation for Ca2+ currents was bell‐shaped, while that for Ba2+, Sr2+, and Na+ currents was sigmoid. Thus depolarisation progressively replaced Ca2+‐dependent inactivation in the fast phase of decay of Ca2+ channel currents with rapid voltage‐dependent inactivation. In the presence of isoproterenol (isoprenaline) the decay of Ca2+ currents was faster than Ba2+, Sr2+ or Na+ currents at all measured voltages (‐40 to +30 mV). The relationship between voltage and inactivation for Ca2+, Ba2+ and Sr2+ currents was bell‐shaped, while that for Na+ currents was sigmoid with less inactivation than under control conditions. Therefore the fast phase of decay of Ca2+ channel currents was now almost entirely due to Ca2+. It is concluded that the relative contributions of Ca2+‐ and voltage‐dependent mechanisms of inactivation of L‐type Ca2+ channels in native cardiac myocytes are modulated by β‐adrenergic stimulation influencing the amount of rapid voltage‐dependent inactivation.

Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein

Ectopic activity in cardiac muscle within pulmonary veins (PVs) is associated with the onset and the maintenance of atrial fibrillation in humans. The mechanism underlying this ectopic activity is unknown. Here we investigate automatic activity generated by catecholaminergic stimulation in the rat PV. Intracellular microelectrodes were used to record electrical activity in isolated strips of rat PV and left atrium (LA). The resting cardiac muscle membrane potential was lower in PV [-70 +/- 1 (SE) mV, n = 8] than in LA (-85 +/- 1 mV, n = 8). No spontaneous activity was recorded in PV or LA under basal conditions. Norepinephrine (10(-5) M) induced first a hyperpolarization (-8 +/- 1 mV in PV, -3 +/- 1 mV in LA, n = 8 for both) then a slowly developing depolarization (+21 +/- 2 mV after 15 min in PV, +1 +/- 2 mV in LA) of the resting membrane potential. Automatic activity occurred only in PV; it was triggered at approximately -50 mV, and it occurred as repetitive bursts of slow action potentials. The diastolic membrane potential increased during a burst and slowly depolarized between bursts. Automatic activity in the PV was blocked by either atenolol or prazosine, and it could be generated with a mixture of cirazoline and isoprenaline. In both tissues, cirazoline (10(-6) M) induced a depolarization (+37 +/- 2 mV in PV, n = 5; +5 +/- 1 mV in LA, n = 5), and isoprenaline (10(-7) M) evoked a hyperpolarization (-11 +/- 3 mV in PV, n = 7; -3 +/- 1 mV in LA, n = 6). The differences in membrane potential and reaction to adrenergic stimulation lead to automatic electrical activity occurring specifically in cardiac muscle in the PV.

Ectopic activity in the rat pulmonary vein can arise from simultaneous activation of α1‐ and β1‐adrenoceptors

Atrial fibrillation (AF) is the most common electrical cardiac disorder in clinical practice. The major trigger for AF is focal ectopic activity of unknown origin in sleeves of cardiac muscle that extend into the pulmonary veins. We examined the role of noradrenaline in the genesis of ectopic activity in the pulmonary vein.

Voltage‐dependent inactivation of l‐type ca2+ Currents in Guinea‐Pig Ventricular Myocytes

The objective of this study was to describe the kinetics of voltage‐dependent inactivation of native cardiac L‐type Ca2+ currents. Whole‐cell currents were recorded from guinea‐pig isolated ventricular myocytes. Voltage‐dependent inactivation was separated from Ca2+‐dependent inactivation by replacing extracellular Ca2+ with Mg2+ and recording outward currents through Ca2+ channels. Voltage‐dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage‐dependent inactivation occurred with τf≈30 ms and τs≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca2+ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time‐independent component of the Ca2+ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca2+ channel current availability at positive membrane potentials and thus a U‐shaped availability‐voltage (A‐V) relationship. It is concluded that β‐adrenergic stimulation altered the reaction of native cardiac L‐type Ca2+ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.

In silico risk assessment for drug-induction of cardiac arrhythmia.

The main components of repolarization reserve for the ventricular action potential (AP) are the rapid (I(Kr)) and slow (I(Ks)) delayed outward K(+) currents. While many drugs block I(Kr) and cause life-threatening arrhythmias including torsades de pointes, the frequency of arrhythmias varies between different I(Kr)-blockers. Different types of block of I(Kr) cause distinct phenotypes of prolongation of action potential duration (APD), increase in transmural dispersion of repolarization (TDR) and, accordingly, occurrence of torsades de pointes. Therefore the assessment of a drugs proarrhythmic risk requires a method that provides quantitative and comprehensive comparison of the effects of different forms of I(Kr)-blockade upon APDs and TDR. However, most currently available methods are not adapted to such an extensive comparison. Here, we introduce I(Kr)-I(Ks) two-dimensional maps of APD and TDR as a novel risk-assessment method. Taking the kinetics of I(Kr)-blockade into account, APDs can be calculated upon a ventricular AP model which systematically alters the magnitudes of I(Kr) and I(Ks). The calculated APDs are then plotted on a map where the x axis represents the conductance of I(Kr) while the y axis represents that of I(Ks). TDR is simulated with models corresponding to APs in epicardial, midcardial and endocardial myocardium. These two-dimensional maps of APD and TDR successfully account for differences in the risk resulting from three distinct types of I(Kr)-blockade which correspond to the effects of dofetilide, quinidine and vesnarinone. This method may be of use to assess the arrhythmogenic risk of various I(Kr)-blockers.

β‐adrenergic and muscarinic agonists modulate inactivation of l‐type ca2+ Channel Currents in Guinea‐Pig Ventricular Myocytes

The objective of this study was to examine the effects of isoproterenol (isoprenaline) and carbachol upon voltage‐dependent inactivation of L‐type Ca2+ current (ICa,L). ICa,L was recorded in guinea‐pig isolated ventricular myocytes in the presence and absence of extracellular Ca2+ to separate total inactivation and voltage‐dependent inactivation. In the presence of Ca2+, isoproterenol and carbachol had ‘competitive’ effects upon the relationships between membrane voltage and ICa,L amplitude and inactivation. Neither agonist had a marked effect upon the decay of inward ICa,L carried by Ca2+. In the absence of Ca2+, isoproterenol severely reduced and slowed ICa,L inactivation; this effect was reversed by carbachol. Under control conditions decay was dominated by fast inactivation. Isoproterenol reduced fast‐inactivating and increased time‐independent currents in a dose‐dependent manner. These effects were counteracted by carbachol. There was a reciprocal relationship between the amplitude of fast‐inactivating and time‐independent currents with agonist stimulation. It is concluded that agonist modulation of rapid voltage‐dependent inactivation of L‐type Ca2+ channels involves an ‘on‐off’ switch.