Antonio Guia

The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca2+ release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca2+ release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.

Ion Concentration-Dependence of Rat Cardiac Unitary L-Type Calcium Channel Conductance

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.

Modulation of the Conductance of Unitary Cardiac L-Type Ca2+ Channels by Conditioning Voltage and Divalent Ions

The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca(2+) channel currents using Ca(2+) and Ba(2+) ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca(2+) channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca(2+) channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba(2+), 18 pS for 5 mM Ba(2+), and 6 pS for 5 mM Ca(2+) ions; these values were 70% greater than the conductance of Ca(2+) channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca(2+) channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca(2+) channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.

Ca2+‐dependent components of inactivation of unitary cardiac L‐type Ca2+ channels

A Ca2+ ion‐dependent inactivation (CDI) of L‐type Ca2+ channels (LCC) is vital in limiting and shaping local Ca2+ ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca2+ ion concentration (2 mm) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble‐averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble‐averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca2+ influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca2+ ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca2+ concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca2+‐dependent signalling processes.

Physiologic gating properties of unitary cardiac L-type Ca2+ channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca(2+) ions through sarcolemmal L-type Ca(2+) channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca(2+) ions permeating the channel. Unitary LCC currents were recorded with 2mM external Ca(2+) ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of -12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53+/-0.02 ms at -30 mV to 1.08+/-0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca(2+) ion entry. Thus, the average amount of Ca(2+) ions entering through an individual LCC opening ( approximately 300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca(2+) ion concentration. Moreover, they provide insight into local Ca(2+)-induced Ca(2+) release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.

NaV1.7 Splice Variant from Human Heart Compared with Neuronal hNaV1.7

Perception of noxious stimuli can be profoundly affected by mutations in the gene SCN9A which encodes the α-subunit of the voltage-gated sodium channel NaV1.7. Mutantions of this channel are associated with chronic pain or complete absence of pain. Primary erythermalgia and paroxysmal extreme pain disorder are syndromes associated with attacks of severe pain resulting from mutations that enhance NaV1.7 channel activity. Non-sense mutations in SCN9A lead to complete loss of NaV1.7 function. Loss of NaV1.7 function produces complete insensitivity to pain and anosmia, but little other changes in functions or behaviors. The pain-specific nature of the mutant NaV1.7 phenotypes is in keeping with the notion that this channel is expressed primarily in dorsal root ganglia and, to a lesser extent in the sympathetic ganglia. These premises make NaV1.7 an ideal target for the development of novel non-addictive analgesics. However, expression of NaV1.7 has been detected in the heart, with 5- to 10-fold higher levels in human cardiac Purkinje fibres versus the right atrium and ventricle and bradycardia and cardiac asystole have been reported in patients with paroxysmal extreme pain disorder. While these events have usually been ascribed to autonomic effects of the NaV1.7 mutations, a more direct effect of altered NaV1.7 in the heart cannot be ruled out. We have set out to clone and characterize the specific NaV1.7 subtype expressed in the human heart. In the present study we cloned and characterized a NaV1.7 subtype predominantly expressed in the human heart. This novel splice variant, missing one exon, may impact drug safety for this emerging family of analgesics. Its biophysical and pharmacological properties have been studied and will be discussed in comparison to the neuronal variant.

Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.