Andre Terzic

Comparative analysis of the kinetic characteristics of L-type calcium channels in cardiac cells of hibernators

An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single Ca2+ channel-conductance and/or keep a fraction of the cardiac L-type Ca2+ channel population in a non-active state.

CHAPTER 9:18O-assisted 31P NMR and Mass Spectrometry for Phosphometabolomic Fingerprinting and Metabolic Monitoring

Comprehensive characterisation of disease-related metabolomic phenotypes and drug effects requires monitoring metabolite levels and their turnover rates. Tandem application of stable isotope 18O-assisted 31P NMR and mass spectrometric techniques uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their dynamics in tissue and blood samples. The 18O labelling procedure is based on incorporation of the 18O atom, provided from H218O, into Pi with each act of ATP hydrolysis and the subsequent distribution of 18O-labelled phosphoryls amongst phosphate-carrying molecules. Essentially, all major phosphometabolites and their turnover rates can be quantified using 18O-assisted 31P NMR spectroscopy and mass spectrometry. This technology permits the simultaneous recording of ATP synthesis and utilisation, phosphotransfer fluxes through adenylate kinase, creatine kinase and glycolytic pathways, as well as mitochondrial nucleotide dynamics and glycogen turnover. Another advantage of 18O methodology is that it can measure almost every phosphotransfer and hydrolytic reaction taking place in the cell including the turnover of small pools of signalling molecules, and the dynamics of energetic signal communication. Our studies demonstrate that 18O-assisted 31P NMR/mass spectrometry is a valuable tool for phosphometabolomic and fluxomic profiling of transgenic models of human diseases, providing valuable data which reveals system-wide adaptations in metabolic networks.

Cardiac ATP-Sensitive Potassium Channel: A Bi-Functional Channel/Enzyme Multimer

Maintenance of myocardial homeostasis critically depends on the ability of cardiac cells to adapt energy-dependent processes in response to metabolic challenge. This requires efficient monitoring of the cellular energetic status, secure delivery of information to energetic sensors and accurate translation of metabolic signals into cellular response. Although advances have been made in resolving the molecular identity of energy-response elements, mechanisms integrating metabolic sensor function with cellular metabolism are largely unknown. Sarcolemmal ATP-sensitive K+ (KATP) channels are prototypic membrane metabolic sensors, which act as bi-functional protein multimers combining catalytic with ion conduction properties. In the hetero-octameric KATP channel complex, the sulfonylurea receptor (SUR2A) harbors an intrinsic ATPase activity and confers fine nucleotide modulation of K+ permeation through the pore-forming Kir6.2 subunit. The metabolic sensor role of KATP channels stems from the non-equivalent properties of nucleotide binding domains (NBD1 and NBD2) in SUR2A. NBD1 binds nucleotides whereas NBD2 hydrolyzes ATP, yet cooperative interaction, rather than the independent contribution of each NBD, is critical for KATP channel regulation. The ATP hydrolysis cycle at SUR2A drives conformational transitions with distinct outcomes on channel gating to adjust membrane potential in response to intracellular metabolic oscillations. Nucleotide exchange between the channel ATPase and the cellular phosphotransfer network provides a mechanistic basis for coupling cell energetics with KATP channel gating. Disruption of phosphotransfer genes produces aberrant metabolic sensing by KATP channels, and generates a cellular phenotype with electrical vulnerability to metabolic challenge. Assigning to the channel catalytic module a role in integrating ion permeation with intracellular enzymatic pathways identifies a novel principle in the regulation of cellular excitability, and defines an operational paradigm for channel/enzyme multimers.