• Introduction
• Major contributions
• Subtypes of low voltage-activated (LVA or T-type) Ca²⁺ channels in central neurons
• Cardiac K⁺ channels in drug-induced pro-arrhythmia
• Membrane ion channels and prostate carcinogenesis
• Our gadgets and innovations

Ion channels of cellular membrane are macromelecular pores providing for passive ion exchange between intra- and extracellular spaces. However, the definition "passive" only implies that specific ion can move through the channel in the direction down, but not up its electrochemical gradient. Otherwise, ion channels are perfectly "active" structures, capable to "decide" when and for how long to open, what type of ion to pass, and what stimulus to respond to. Since ions are charged particles, when moving through the channel they create electrical current, which changes membrane potential. Consequently, the role of ion channels in electrical signaling within the cell and among the cells - phenomena generally known as electrical excitability - was first to be recognized. However, since then, it became evident that there is basically no normal or pathologic cellular function that ion channels would not be involved in.

There are lots of different types of ion channels, which are generally classified according to ion(s) they transport or stimulus that gates them. They are differentially expressed in various cells to best suit specific cellular function(s). As they pass electrical current, the best way to study them is to measure this current by means of electrophysiological techniques. Recent widespread application of molecular biology to ion channels allowed cloning of many their representatives and study structure-function relationships in the channel's molecule outside its native environment in the foreign expression systems (heterologous expression). Whatever ion channel is investigated, the principal questions, which are usually asked, are: (i) what are the functional properties of the channel? (ii) what physiological and/or pathological processes it is involved in? (iii) what molecular determinant(s) are responsible for specific properties of the channel? and (iv) what pharmacological or other means can be useful for influencing channel function in the desired direction via affecting these determinants?

Our laboratory has a longstanding interest in various aspects of ion channels function, structure, regulation and involvement in pathological states. Since the beginning of 80-s we contributed to a number of important findings in the field. These contributions provided the basis for pursuing three current projects:

1. Subtypes of low voltage-activated (LVA or T-type) Ca²⁺ channels in central neurons.
2. Cardiac K⁺ channels in drug-induced pro-arrhythmia.
3. Membrane ion channels and prostate carcinogenesis.

Since the collapse of the Soviet Union the situation with science funding considerably deteriorated in Ukraine. Therefore, for supporting our research we have to rely mostly on collaborative grants with Western partners, which are provided by International granting agencies on competitive basis. Every initiative for such collaborative project from interested Western parties is welcomed.

Essentially contributed to the discovery of the phenomenon of Ca²⁺-dependent regulation of Ca²⁺-channel selectivity and permeation (J. Membr .Biol., 1983, 76:83-93), description of neuronal Ca²⁺ channels diversity (Pflügers Arch., 1988, 411:661-669), characterization of low voltage-activated (LVA) Ca²⁺ conductance in general (J. Physiol., 1991, 443:25-44) and in central neurons in particular (Neurosci., 1996, 70:729-738, Brain Res., 1998, 783:280-285, Neuroreport, 1999, 10:651-652; Pflügers Arch., 2001, 441:832-839), developing of a four-state kinetic model for Ca²⁺ channel activation (J. Membr. Biol., 1989, 110:29-38, J. Physiol., 1991, 443:25-44), assessment of a direct, Gs protein-mediated pathway in β-adrenergic regulation of cardiac Ca²⁺ and Cl^- channels (J. Physiol., 1990, 424:205-228, Am. J. Physiol., 1990, 259:H264-H267, Am. J. Physiol., 1997, 273:H2539-H2548), characterization of β-adrenergic regulation of cardiac Na⁺-Ca²⁺ exchanger (Proc. Natl. Acad. Sci. USA, 1996, 93:5527-5532, J. Biol. Chem., 1998, 273:18819-18825), evaluation of pharmacological and hormonal regulation of cardiac K⁺ channels (J. Cardiovasc. Pharm., 1997, 30:42-45, J. Pharm. Exp. Ther., 1997, 281:233-244, Biochem. Pharm., 2001, 62:41-49), characterization of membrane conductances in prostate cancer epithelial cells (Am. J. Physiol., 2000, 279:C1144-1154, J. Physiol. 2000, 527:71-83, FASEB J., 2002, 16:222-224, Cancer Cell, 2002, 1:169-179, J. Bioenerg. Biomembr., 2002, 34:307-315) and glial cells (Glia, 2003, 42:325-339, Neuroreport, 2004, 15:321-324).

During Soviet era all research has been conducted at Bogomoletz Institute of Physiology (BIPh), National Academy of Sciences of Ukrainian (NASU), Kyiv, Ukraine. However, after collapse of the Soviet Union essential part of many projects has been performed on collaborative basis on the sites of our foreign collaborators at II Physiologisches Institut, Universitat des Saarlandes (Homburg/Saar, Germany, Prof. W. Trautwein, Dr. D. Pelzer), Department of Physiology and Biophysics, Dalhousie University (Halifax, Canada, Prof. T. McDonald, Prof. D. Pelzer), Department of Pharmacology, Georgetown University (Washington, DC, USA, Prof. M. Morad, Prof. R. Woosley), Laboratoire de Physiologie Cellulaire, Universite des Sciences et Technologies de Lille (Villeneuve d'Ascq, France, Prof. N. Prevarskaya, Prof. R. Skryma), Department of Neurosurgery, University of Maryland (Baltimore, USA, Prof. M. Simard, Dr. V. Gerzanich), Department of Biochemistry, Universidad Central del Caribe (Bayamon, Puerto Rico, Dr. S. Skatchkov, Dr. M. Eaton).

LVA (or T-type) Ca²⁺ channels represent a subfamily of voltage-gated Ca²⁺-selective ion channels with voltage-dependent properties that permit their activation only from very negative membrane potentials (<-80 mV). Due to this property the role of LVA Ca²⁺ channels in electrical excitability is mainly related to generation of spontaneous membrane potential fluctuations around resting potential, pacemaking and bursting spiking. In addition, by transporting Ca²⁺ ions they are also involved in a number of traditional Ca²⁺-dependent processes, in which Ca²⁺ ions play role of second messengers (e.g. hormone secretion, smooth muscle contraction, fertilization, cell growth and differentiation, etc.). LVA Ca²⁺ channels are ubiquitously expressed in various cells and tissues, where they, although sharing the common feature of low voltage of activation, nevertheless display quite variable other functional properties, suggesting the existence of their several subtypes. Recent cloning of three primary pore-forming T-channel α1-subunits, α1G (Cav3.1), α1H (Cav3.2) and α1I (Cav3.3) (Perez-Reyes et al., Nature, 1998, 56:660-669, Cribbs et al., Circ. Res., 1998, 83:103-109, Lee et al., J. Neurosci., 1999, 19:1012-1921), provided structural basis for functional diversity.

LVA Ca²⁺ channel-mediated signaling is of special significance in the brain structures, which are known to be involved in rhythmogenesis, like the thalamic nuclei. The generation of burst responses by thalamic neurons is Ca²⁺-dependent, requiring conditioning steady or transient membrane hyperpolarization and thus indicating the involvement of LVA Ca²⁺ channels in the mechanism of their generation. Consistent with such involvement, various types of thalamic neurons express high levels of LVA Ca²⁺ current, which together with appropriate combination of excitatory and inhibitory inputs can lead to the rebound excitation and eventually result in the maintenance of synchronized, repetitive burst firing typical, for instance, to certain stages of sleep. Overexpression of LVA Ca²⁺ current can also be the reason for the hypersynchronous neuronal activity that is likely to contribute to the generalized absence epilepsy.

Our own functional studies conducted on neurons of the laterodorsal (LD) thalamic nucleus of rat suggested that these neurons express at least two functional subtypes of LVA Ca²⁺ channels, which because of the distinct kinetics of inactivation were tentatively termed as "fast" and "slow". These channel subtypes differed not only in their voltage-dependence, kinetics, pharmacology and selectivity (Neuroreport, 1999, 10:651-657; Pflügers Arch., 2001, 441:832-839), but also in ontogenic expression (Tarasenko et al., J. Physiol., 1997, 499:77-86), possible localization on the neuron body and physiological roles (Pflügers Arch., 2001, 441:832-839). Our current research is aimed at correlating endogenous thalamic LVA-channel subtypes at various developmental stages of the animal to the specific recombinant T-channel α1-subunits. To do so we use functional criteria, especially comparison of pharmacology of endogenous channels and heterologously expressed α1-subunits, as well as juxtapose LVA current profiles with mRNA levels for specific α1-subunits in thalamic neurons. This research is backed by generous gift of T-type Ca²⁺-channel clones from the pioneer in their cloning Dr. E. Perez-Reyes (University of Virginia).

The QT interval of the electrocardiogram (ECG) reflects the length of time during which the ventricles are depolarized and therefore serves as a measure of cardiac ventricular action potential duration. Significant prolongation of the QT interval, the so-called "long QT syndrome" (LQTS), is associated with delayed cardiac repolarization, and is a primary cause of a polymorphic ventricular arrhythmia known as torsades de pointes (TdP) that can degenerate into ventricular fibrillation, leading to sudden death in otherwise healthy individuals. LQTS can be either inherited or acquired. The former is associated with inherited ion channel defects, whilst the later most often results from administration of structurally diverse drugs that can block cardiac K⁺ channels, in particular channels carrying the rapid delayed rectifier current, IKr (J. Cardiovasc. Pharm., 1997, 30:42-45; J. Pharm. Exp. Ther., 1997, 281:233-244).

Clinical observations and experimental evidence from a number of laboratories have demonstrated unequivocally that female gender is associated with a significantly higher risk of developing drug-induced TdP than is male gender. The precise mechanism(s) for the increased risk in women is/are unknown, but may be related to the fact that the baseline QT interval is naturally longer in women compared to men, suggesting that sex hormones may modulate cardiac K⁺ channels function, expression and interaction with pro-arrhythmic agents.

The pore-forming subunit of the channel that carries IKr is encoded by Human Ether a-go-go-Related Gene (HERG). In our recent study we have shown that testosterone is able not only to modulate expression of HERG channel in heterologous system, but also to influence the characteristics of its blockade by the drugs with reported incidences of pro-arrhythmic side effects in clinical use (Shuba et al., Biochem. Pharmacol., 2001, 62:41-49). These results suggest protective role of testosterone against drug-induced arrhythmogenesis and, therefore, are of potentially great practical importance. However, their direct extrapolation to mammalian cardiac tissue is still premature, as they were obtained in Xenopus oocyte expression system, which is characterized by its own, quite specific features of hormonal action. As hetereologously expressed channels provide a number of experimental advantages over endogenous ones in native cardiac myocytes, we are currently focusing on reproducing our main findings on hormone-drug-channel interactions in the expression system most closely resembling native cardiomyocytes and studying intimate mechanisms involved.

Prostate cancer (PC) is one of the leading threats to men's health. Its early stage depends on androgens for growth and survival, and androgen ablation therapy may at this time be effective in causing tumor to regress, however, in the late, androgen-independent stage there is currently no successful therapy. Androgen-independence is associated with the appearance of new cell phenotypes, characterized by apoptosis (programmed cell death) inhibition rather than enhanced proliferation. It is, therefore, vital to understand what drives the progression to androgen independence.

Until recently, the problems of normal and pathological development of the human prostate have mainly been addressed from biochemical and molecular biology perspectives, whereas the role of Ca²⁺ signaling and even more so plasma membrane ion channels have been largely ignored. At the same time the role of Ca²⁺ entry pathways in cell-cycle progression, control of cell proliferation and onset of apoptosis - the processes balance or imbalance among which distinguishes normal development from uncontrolled growth - is well recognized. The same processes are also associated with essential perturbations in volume homeostasis involving activity of other types of ion channels, particularly K⁺ and Cl^- ones. Regulation of prostate growth and secretory activity by endocrine means and autonomic nervous system also necessarily involves activation of membrane ion channels.

In the framework of current project, which is collaborative initiative with the Laboratoire de Physiologie Cellulaire headed by Prof. N. Prevarskaya in the Universite des Sciences et Technologies de Lille (France), we investigate ion channels responsible for Ca²⁺ entry and regulatory volume decrease (RVD) in prostate cancer epithelial cells. In particular, we are interested in the role of various members of TRP (Transient Receptor Potential) channel family in agonist-induced store-dependent and store-independent Ca²⁺ entry and volume-regulated anion channels (VRAC) in RVD. We also try to establish how Ca²⁺ entry pathways and RVD evolve during acquisition of androgen-independence and apoptotic resistance characteristic of advanced prostate cancer, which is important for establishing new therapeutic strategies.

• The system for solutions application to Xenopus oocytes
  
  
• Temperature- and concentration-clamp system for rapid solution exchange around patch-clamped cell
  
  
• Miniature valve that can be placed anywhere