Heterogeneity and function of K(ATP) channels in canine hearts.

BACKGROUND: The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels. OBJECTIVE: To assess SUR subunit composition of K(ATP) channels and consequence of K(ATP) activation for action potential duration (APD) in dog hearts. METHODS: Patch-clamp techniques were used on isolated dog cardiomyocytes to investigate K(ATP) channel properties. Dynamic current clamp, by injection of a linear K(+) conductance to simulate activation of the native current, was used to study the consequences of K(ATP) activation on APD. RESULTS: Metabolic inhibitor (MI)-activated current was not significantly different from pinacidil (SUR2A-specific)-activated current, and both currents were larger than diazoxide (SUR1-specific)-activated current in both the atrium and the ventricle. Mean K(ATP) conductance (activated by MI) did not differ significantly between chambers, although, within the ventricle, both MI-induced and pinacidil-induced currents tended to decrease from the epicardium to the endocardium. Dynamic current-clamp results indicate that myocytes with longer baseline APDs are more susceptible to injected K(ATP) current, a result reproduced in silico by using a canine action potential model (Hund-Rudy) to simulate epicardial and endocardial myocytes. CONCLUSIONS: Even a small fraction of K(ATP) activation significantly shortens APD in a manner that depends on existing heterogeneity in K(ATP) current and APD.

Impact of differential right-to-left shunting on systemic perfusion in pulmonary arterial hypertension.

OBJECTIVES: This study aimed at identifying the ideal right-to-left shunt-fraction to improve cardiac output (CO) and systemic perfusion in pulmonary arterial hypertension (PHT). BACKGROUND: Atrial septostomy (AS) has been a high-risk therapeutic option for symptomatic drug-refractory patients with PHT. Results have been unpredictable due to limited knowledge of the optimal shunt-quantity. METHODS: In nine dogs, an 8-mm shunt-prosthesis was inserted between the superior vena cava (SVC) and the left atrium. With pulmonary artery (PA) banding, mean (± SEM) systolic right ventricular pressure increased from 37 ± 1 mm Hg at baseline to 44 ± 1 mm Hg (moderate PHT, P = 0.005) and 50 ± 2 mm Hg (severe PHT, P < 0.001). Shunt-flow was adjusted by total (forcing all flow through the shunt) or partial occlusion of the SVC and partial or total clamping of the shunt. Caval-, shunt-, and aortic-flow were measured by ultrasonic flow-probes. Blood gases were drawn from the aortic root and PA. RESULTS: At severe PHT, a shunt-flow of 11 ± 1% of CO (253 ± 90 mL/min) increased CO significantly by 25% (1.8 ± 0.1 to 2.4 ± 0.2 L/min, P = 0.005) causing an increase of systemic oxygen delivery index (DO2 I) by 23% (309 ± 23 to 399 ± 32 mL/min/m(2), P = 0.035). Arterial O2 -saturation did not change significantly until a shunt-flow of 18 ± 2% was exceeded, causing a drop from 96 ± 1% to 84 ± 4% (P = 0.013). At moderate PHT, CO or DO2 I did not improve significantly at any shunt-flow. CONCLUSIONS: In severe PHT, a shunt-flow of 11% of CO represented the ideal shunt-fraction. Augmentation of CO compensated for declined O2 -saturation due to right-to-left shunting and improved DO2 I. In moderate PHT, AS is less promising.

Differential impact of short periods of rapid atrial pacing on left and right atrial mechanical function.

Current techniques to describe atrial function are limited by their load dependency and hence do not accurately reflect intrinsic mechanical properties. To assess the impact of atrial fibrillation on atrial function, combined pressure-volume relationships (PVR) measured by conductance catheters were used to evaluate the right (RA) and left (LA) atrium in 12 isoflurane-anesthetized pigs. Biatrial PVR were recorded over a wide range of volumes during transient caval occlusion at baseline sinus rhythm (SR), after onset of rapid atrial pacing (RAP), after 1 h of RAP, after conversion to SR, and after 1 h of recovery. Cardiac output decreased by 16% (P = 0.008) with onset of RAP. Mean LA and RA pressures increased by 21 and 40% (P < 0.001), respectively, and remained elevated during the entire recovery period. RA reservoir function increased from 51 to 58% and significantly dropped to 43% after resumption of SR (P = 0.017). Immediately after RAP, a right shift of LA end-systolic PVR-intercept for end-systolic volume required to generate an atrial end-systolic pressure of 10 mmHg (24.4 ± 4.9 to 28.1 ± 5.2 ml, P = 0.005) indicated impaired contractility compared with baseline. Active LA emptying fraction dropped from 17.6 ± 7.5 to 11.7 ± 3.7% (P < 0.001), LA stroke volume and ΔP/Δt(max)/P declined by 22% (P = 0.038 and 0.026, respectively), while there was only a trend to impaired RA systolic function. Stiffness quantified by the ratio of pressure to volume at end-diastole was increased immediately after RAP only in the RA (P = 0.020), but end-diastolic PVR shifted rightward in both atria (P = 0.011 LA, P = 0.045 RA). These data suggest that even short periods of RAP have a differential impact on RA and LA function, which was sustained for 1 h after conversion to SR.

Patch clamp and perfusion techniques for studying ion channels expressed in Xenopus oocytes.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release. BK channels are activated by membrane depolarization and by intracellular Ca(2+) and Mg(2+). Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18°C. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Modulation of BK channel gating by the ß2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1.

Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory ß subunits. Four types of BK channel ß subunits (ß1-ß4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the ß1 and ß2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these ß subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for ß2 modulation based on the study of differential effects of the ß2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (ßA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the ß2 but not for the ß1 modulation. These results suggest that the ß2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel ß subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.

BK channel activation: structural and functional insights.

The voltage- and Ca(2+)-activated K(+) (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K(+) channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first glimpse into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated.

{beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia.

Large conductance Ca(2+)-activated K(+) (BK) channels modulate many physiological processes including neuronal excitability, synaptic transmission and regulation of myogenic tone. A gain-of-function (E/D) mutation in the pore-forming alpha subunit (Slo1) of the BK channel was recently identified and is linked to human neurological diseases of coexistent generalized epilepsy and paroxysmal dyskinesia. Here we performed macroscopic current recordings to examine the effects of the E/D mutation on the gating kinetics, and voltage and Ca(2+) dependence of the BK channel activation in the presence of four different beta subunits (beta1-4). These beta subunits are expressed in a tissue-specific pattern and modulate BK channel function differently, providing diversity and specificity for BK channels in various physiological processes. Our results show that in human (h) Slo1-only channels, the E/D mutation increased the rate of opening and decreased the rate of closing, allowing a greater number of channels to open at more negative potentials both in the presence and absence of Ca(2+) due to increased Ca(2+) affinity and enhanced activation compared with the wild-type channels. Even in the presence of beta subunits, the E/D mutation exhibited these changes with the exception of beta3b, where Ca(2+) sensitivity changed little. However, quantitative examination of these changes shows the diversity of each beta subunit and the differential modulation of these subunits by the E/D mutation. For example, in the presence of the beta1 subunit the E/D mutation increased Ca(2+) sensitivity less but enhanced channel activation in the absence of Ca(2+) more than in hSlo1-only channels, while in the presence of the beta2 subunit the E/D mutation also altered inactivation properties. These findings suggest that depending on the distribution of the various beta subunits in the brain, the E/D mutation can modulate BK channels differently to contribute to the pathophysiology of epilepsy and dyskinesia. Additionally, these results also have implications on physiological processes in tissues other than the brain where BK channels play an important role.

Subunit-specific effect of the voltage sensor domain on Ca2+ sensitivity of BK channels.

Large conductance Ca(2+)- and voltage-activated K(+) (BK) channels, composed of pore-forming alpha-subunits and auxiliary beta-subunits, play important roles in diverse physiological processes. The differences in BK channel phenotypes are primarily due to the tissue-specific expression of beta-subunits (beta1-beta4) that modulate channel function differently. Yet, the molecular basis of the subunit-specific regulation is not clear. In our study, we demonstrate that perturbation of the voltage sensor in BK channels by mutations selectively disrupts the ability of the beta1-subunit--but not that of the beta2-subunit--to enhance apparent Ca(2+) sensitivity. These mutations change the number of equivalent gating charges, the voltage dependence of voltage sensor movements, the open-close equilibrium of the channel, and the allosteric coupling between voltage sensor movements and channel opening to various degrees, indicating that they alter the conformation and movements of the voltage sensor and the activation gate. Similarly, the ability of the beta1-subunit to enhance apparent Ca(2+) sensitivity is diminished to various degrees, correlating quantitatively with the shift of voltage dependence of voltage sensor movements. In contrast, none of these mutations significantly reduces the ability of the beta2-subunit to enhance Ca(2+) sensitivity. These results suggest that the beta1-subunit enhances Ca(2+) sensitivity by altering the conformation and movements of the voltage sensor, whereas the similar function of the beta2-subunit is governed by a distinct mechanism.