P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to Kv channels, but show much lower selectivity for K+. The ∼3.3 Å selectivity filter of K+ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L478C479IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K+ channels, we also studied C479T channels. Based on permeability ratios (PX/PK), C479T HCN4 channels (K+(1)>Rb+(0.85)>Cs+(0.59)>Li+(0.50)≥Na+(0.49)) were less selective than WT rabbit HCN4 (K+(1)>Rb+(0.48)>Cs+(0.31)≥Na+(0.29)>Li+(0.03)), indicating that the TIGYG sequence is insufficient to confer K+ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K+ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K+ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K+ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K+ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K+ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K+ channels originate from differences outside the P-loop region

Changes in Heart Rate and Its Regulation by the Autonomic Nervous System Do Not Differ Between Forced and Voluntary Exercise in Mice

Most exercise studies in mice have relied on forced training which can introduce psychological stress. Consequently, the utility of mouse models for understanding exercise-mediated effects in humans, particularly autonomic nervous system (ANS) remodeling, have been challenged. We compared the effects of voluntary free-wheel running vs. non-voluntary swimming on heart function in mice with a focus on the regulation of heart rate (HR) by the ANS. Under conditions where the total excess O2 consumption associated with exercise was comparable, the two exercise models led to similar improvements in ventricular function as well as comparable reductions in HR and its control by parasympathetic nervous activity (PNA) and sympathetic nervous activity (SNA), compared to sedentary mice. Both exercise models also increased HR variability (HRV) by similar amounts, independent of HR reductions. In all mice, HRV depended primarily on PNA, with SNA weakly affecting HRV at low frequencies. The differences in both HR and HRV between exercised vs. sedentary mice were eliminated by autonomic blockade, consistent with the similar intrinsic beating rates observed in atria isolated from exercised vs. sedentary mice. In conclusion, both forced and voluntary exercise induce comparable ventricular physiological remodeling as well as HR reductions and HR-independent enhancements of HRV which were both primarily dependent on increased PNA.New and noteworthy–No previous mouse studies have compared the effects of forced and voluntary exercise on the heart function and its modulation by the autonomic nervous system (ANS).–Both voluntary free-wheel running and forced swimming induced similar improvements in ventricular contractile function, reductions in heart rate (HR) and enhancements of HR variability (HRV).–HR regulation in exercised mice was linked to increased parasympathetic nerve activity and reduced sympathetic nerve activity.– HRV was independent of HR and depended primarily on PNA in both exercised and sedentary mice.– Complete cardiac autonomic blockade eliminated differences in both HR and HRV between exercised and sedentary mice

Conduction through the Inward Rectifier Potassium Channel, Kir2.1, Is Increased by Negatively Charged Extracellular Residues

Ion channel conductance can be influenced by electrostatic effects originating from fixed “surface” charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6–293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of γ on the K+ activity (aK) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as aK approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-d-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the γ–aK relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic γ–aK relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge

Activated plasma coagulation β-Factor XII-induced vasoconstriction in rats

By inducing BK (bradykinin)-stimulated adrenomedullary catecholamine release, bolus injection of the β-fragment of activated plasma coagulation Factor XII (β-FXIIa) transiently elevates BP (blood pressure) and HR (heart rate) of anaesthetized, vagotomized, ganglion-blocked, captopril-treated bioassay rats. We hypothesized that intravenous infusion of β-FXIIa into intact untreated rats would elicit a qualitatively similar vasoconstrictor response. BN (Brown Norway) rats received for 60 min either: (i) saline (control; n=10); (ii) β-FXIIa (85 ng/min per kg of body weight; n=9); or (iii) β-FXIIa after 2ADX (bilateral adrenalectomy; n=9). LV (left ventricular) volume and aortic BP were recorded before (30 min baseline), during (60 min) and after (30 min recovery) the infusion. TPR (total peripheral resistance) was derived from MAP (mean arterial pressure), SV (stroke volume) and HR. Saline had no haemodynamic effects. β-FXIIa infusion increased its plasma concentration 3-fold in both groups. In adrenally intact rats, β-FXIIa infusion increased MAP by 6% (5±2 mmHg) and TPR by 45% (0.50±0.12 mmHg/ml per min), despite falls in SV (−38±8 μl) and HR [−18±5 b.p.m. (beats/min)] (all P<0.05). In 2ADX rats, β-FXIIa had no HR effect, but decreased SV (−89±9 μl) and MAP (−4±1 mmHg), and increased TPR by 66% (0.59±0.15 mmHg/ml per min) (all P<0.05). After infusion, adrenally intact rats exhibited persistent vasoconstriction (MAP, 10±1 mmHg; TPR, 0.55±0.07 mmHg/ml per min; both P<0.05), whereas in 2ADX rats, MAP remained 5±1 mmHg below baseline (P<0.05) and TPR returned to baseline. End-study arterial adrenaline (epinephrine) concentrations in the three groups were 1.9±0.6, 9.8±4.1 and 0.6±0.2 nmol/l respectively. Thus, in neurally intact lightly anaesthetized untreated rats, β-FXIIa infusion induces both adrenal catecholamine-mediated and adrenally independent increases in peripheral resistance

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated

The Force-sarcomere relation in cardiac myocardium

Bibliography: p. 319-347