Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings

Modelling and simulation studies of potassium channels

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Filter Flexibility in a Mammalian K Channel: Models and Simulations of Kir6.2 Mutants

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that ∼50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels

Diagnostic and prognostic significance of cardiovascular magnetic resonance native myocardial T1 mapping in patients with pulmonary hypertension

Background: Native T1 may be a sensitive, contrast-free, non-invasive cardiovascular magnetic resonance (CMR) marker of myocardial tissue changes in patients with pulmonary artery hypertension. However, the diagnostic and prognostic value of native T1 mapping in this patient group has not been fully explored. The aim of this work was to determine whether elevation of native T1 in myocardial tissue in pulmonary hypertension: (a) varies according to pulmonary hypertension subtype; (b) has prognostic value and (c) is associated with ventricular function and interaction. Methods: Data were retrospectively collected from a total of 490 consecutive patients during their clinical 1.5 T CMR assessment at a pulmonary hypertension referral centre in 2015. Three hundred sixty-nine patients had pulmonary hypertension [58 ± 15 years; 66% female], an additional 39 had pulmonary hypertension due to left heart disease [68 ± 13 years; 60% female], 82 patients did not have pulmonary hypertension [55 ± 18; 68% female]. Twenty five healthy subjects were also recruited [58 ±4 years); 51% female]. T1 mapping was performed with a MOdified Look-Locker Inversion Recovery (MOLLI) sequence. T1 prognostic value in patients with pulmonary arterial hypertension was assessed using multivariate Cox proportional hazards regression analysis. Results: Patients with pulmonary artery hypertension had elevated T1 in the right ventricular (RV) insertion point (pulmonary hypertension patients: T1 = 1060 ± 90 ms; No pulmonary hypertension patients: T1 = 1020 ± 80 ms p < 0.001; healthy subjects T1 = 940 ± 50 ms p < 0.001) with no significant difference between the major pulmonary hypertension subtypes. The RV insertion point was the most successful T1 region for discriminating patients with pulmonary hypertension from healthy subjects (area under the curve = 0.863) however it could not accurately discriminate between patients with and without pulmonary hypertension (area under the curve = 0.654). T1 metrics did not contribute to prediction of overall mortality (septal: p = 0.552; RV insertion point: p = 0.688; left ventricular free wall: p = 0.258). Systolic interventricular septal angle was a significant predictor of T1 in patients with pulmonary hypertension (p < 0.001). Conclusions: Elevated myocardial native T1 was found to a similar extent in pulmonary hypertension patient subgroups and is independently associated with increased interventricular septal angle. Native T1 mapping may not be of additive value in the diagnostic or prognostic evaluation of patients with pulmonary artery hypertension

Diagnostic and prognostic significance of cardiovascular magnetic resonance native myocardial T1 mapping in patients with pulmonary hypertension

Abstract Background Native T1 may be a sensitive, contrast-free, non-invasive cardiovascular magnetic resonance (CMR) marker of myocardial tissue changes in patients with pulmonary artery hypertension. However, the diagnostic and prognostic value of native T1 mapping in this patient group has not been fully explored. The aim of this work was to determine whether elevation of native T1 in myocardial tissue in pulmonary hypertension: (a) varies according to pulmonary hypertension subtype; (b) has prognostic value and (c) is associated with ventricular function and interaction. Methods Data were retrospectively collected from a total of 490 consecutive patients during their clinical 1.5 T CMR assessment at a pulmonary hypertension referral centre in 2015. Three hundred sixty-nine patients had pulmonary hypertension [58 ± 15 years; 66% female], an additional 39 had pulmonary hypertension due to left heart disease [68 ± 13 years; 60% female], 82 patients did not have pulmonary hypertension [55 ± 18; 68% female]. Twenty five healthy subjects were also recruited [58 ±4 years); 51% female]. T1 mapping was performed with a MOdified Look-Locker Inversion Recovery (MOLLI) sequence. T1 prognostic value in patients with pulmonary arterial hypertension was assessed using multivariate Cox proportional hazards regression analysis. Results Patients with pulmonary artery hypertension had elevated T1 in the right ventricular (RV) insertion point (pulmonary hypertension patients: T1 = 1060 ± 90 ms; No pulmonary hypertension patients: T1 = 1020 ± 80 ms p < 0.001; healthy subjects T1 = 940 ± 50 ms p < 0.001) with no significant difference between the major pulmonary hypertension subtypes. The RV insertion point was the most successful T1 region for discriminating patients with pulmonary hypertension from healthy subjects (area under the curve = 0.863) however it could not accurately discriminate between patients with and without pulmonary hypertension (area under the curve = 0.654). T1 metrics did not contribute to prediction of overall mortality (septal: p = 0.552; RV insertion point: p = 0.688; left ventricular free wall: p = 0.258). Systolic interventricular septal angle was a significant predictor of T1 in patients with pulmonary hypertension (p < 0.001). Conclusions Elevated myocardial native T1 was found to a similar extent in pulmonary hypertension patient subgroups and is independently associated with increased interventricular septal angle. Native T1 mapping may not be of additive value in the diagnostic or prognostic evaluation of patients with pulmonary artery hypertension