Synthetic and molecular engineering of Ca2+ signaling
Toward this end, our lab exploits a multidisciplinary approach. First, we use the "patch-clamp technique" to open a window into the molecular operation of ion channels by allowing us to examine the stochastic function of one, or a few such channel molecule(s) at a time. As well, the patch-clamp method enables examination of the "gating currents" arising from the very movement of voltage-sensors that activate voltage-gated ion channels. Second, we use the techniques of molecular biology to express cloned calcium channels, and to selectively mutate or make chimeric constructs of the channels to be expressed. When combined with the other strategies, this capability permits structure-function correlations to be made about the molecular mechanism of channel operation. Third, we are developing new optical imaging and sensor technology to detect protein-protein interaction in living cells, based on fluorescence energy transfer between engineered green fluorescent proteins. Fourth, we use novel adenoviral techniques, with inducible promoters, to express wild-type or altered calcium channel genes in primary cultures of heart cells and neurons. This paradigm permits exploration of the biological role of certain channel phenotypes in cellular physiology. Also, when channel subunits are tagged with green fluorescent protein (GFP), we can investigate the time-dependent spatial dispersion of channels. Our viral gene-expression approach provides a rapid complement to experiments on transgenic animals, ones which can take years to complete. Fifth, where possible, our lab emphasizes mathematical formulation, analysis, and modelling to interpret, refine, and design experiments.
Together, these approaches allow us to test hypotheses regarding the structure-function relations of channels, to determine why certain phenotypic properties of calcium channels are important to cellular function, and to develop novel technologies for approaching these sorts of questions. The focus area of our research provides a remarkable opportunity for the fruitful combination of mathematics, engineering, and molecular experimentation. In this important biological setting, the richness of the data are well-matched to rigorous quantitative analysis. Main projects taken from our overall research thrust are listed below.
Calcium channel inactivation: Inactivation of calcium channels by repetitive activation figures critically in control of the heartbeat and neuronal signal processing. There are two fundamentally different types of inactivation.
In the first, L-type calcium channels (or "C-class" channels) inactivate in response to local elevations of intracellular [Ca2+], providing an important form of negative feedback in many cell types. Both the functional properties and molecular mechanisms of this important type of feedback have remained elusive for more than a decade. Using extensive single-channel recording and novel biophysical analysis, we have made inroads into the functional basis for Ca2+ inactivation (Imredy and Yue, 1994), leading to the development of detailed mathematical models of this type of inactivation. We have also begun to understand the structural basis for Ca2+ inactivation. We took advantage of the fact that not all classes of calcium channels have Ca2+ inactivation (e.g., so-called "E-class" channels lack the inactivating phenotype), despite a large degree of structural homology. We thus constructed a series of chimeric channels composed of portions of C- and E-class channels. In so doing, we identified a consensus Ca2+-binding motif (an "EF hand") that is essential for the Ca2+ inactivation process (de Leon et al., 1995). We are continuing to refine our understanding of the precise molecular determinants of Ca2+ inactivation embodied in this region of C-class channels (Peterson et al., 1999 abstract), to explore the role of collaborating proteins in this form of inactivation, and to develop a structural mechanism for the inactivation process.
Second, neuronal P/Q-, N-, and R-type channels inactivate profoundly according to a voltage-dependent mechanism. We have recently found that although these channels inactivate slowly during square-pulse depolarization, they inactivate profoundly during a train of neuronal action-potential waveforms (Patil et al., 1998), with potentially important neurophysiological implications. The underlying mechanism turns out to be unusual by comparison to classic mechanisms for voltage-gated Na and K channels. These calcium channels inactivate preferentially from intermediate closed states along the activation pathway, allowing inactivation to extend beyond the brief duration of action-potential waveforms to continue between spikes, as the channel undergoes repetitive cycles of activation and deactivation. Mathematical simulations of this sort of mechanism successfully explain these unusual performance features. Ongoing work seeks to refine quantitative gating models of such "preferential closed-state inactivation," to understand its structural basis, and to investigate its role in actual neuronal preparations (see section on "Role of calcium channels in heart and nerve," below).
G-protein modulation of neuronal calcium channels. Little in-depth knowledge is available about the opening and closing properties ("gating") of neuronal, as opposed to cardiac, calcium channels. Part of the difficulty has been the coexistence in neurons of multiple forms of neuronal calcium channels, making it difficult to attribute observed properties to one type of channel. We have taken the approach of characterizing heterologously expressed neuronal calcium channels, expressed one-at-a-time from isolated cDNA clones. With assurance of the channel type being expressed, we are characterizing the gating and modulation of three major forms of neuronal calcium channels, A-class (so-called P/Q-type channels), B-class (or N-type channels), and E-class (or R-type). In regard to A- and B-class channels, we have developed a recombinant system in which to study G-protein regulation of these channels, a form of regulation that plays a key role in the modulation of synaptic strength. This model system for studying G-protein modulation is permits application of a number of experimental techniques to understand the structural mechanisms underlying G-protein modulation. These include: (1) high-resolution single-channel recording using quartz pipette technnology; (2) gating-current measurements, and (3) novel optical methods using fluorescence resonance energy transfer (FRET). Overall, this system provides an exciting opportunity to understand the functional and structural basis for differences in gating and modulation of the different types of neuronal calcium channels (Patil et al., 1996; Brody et al., 1997, Jones et al., 1997, Jones et al., 1998, Colecraft et al., 1998 and 1999 abstracts).
Calcium-channel permeation and block by pharmaceutical drugs: The pore region of calcium channels is crucial to the fundamental mechanism whereby Ca2+ passes through channels so quickly and selectively. This region is also critical to drug interaction with calcium channels. We are interested in the relation of the channel pore to both of these roles.
In regard to drug interaction, use-dependence is one of the sought-after properties of pharmaceutical blockers of ion channels. A drug has use-dependence if a channel is increasingly modulated by a drug the more frequently the channel is activated by voltage. In the setting of cardiac arrhythmias or seizures, for example, use-dependence is beneficial because channels in healthy tissue are not appreciably inhibited, but those involved in pathologically rapid stimulation are blocked. The molecular basis of use-dependence remains unknown. Recently, we have found that two chemical classes of calcium channel blocking compounds behave differently in respect to their block of the various classes of recombinant calcium channels. The phenylalkylamines block A-, C-, and E-class channels in a use-dependent manner. In striking contrast, the benzothiazipines block only the C-class in a use-dependent manner. A- and E- class are blocked in a use-independent manner. Chimeras of C and E class channels have been constructed to gain insight into the structural determinants of use-dependence for the benzothiazipine class. The detailed understanding of use-dependence of benzothiazipines in calcium channels may provide general insight into this phenomena in the setting of other ionic channels and drugs (Cai et al., 1997).
Regarding permeation, different molecular classes of calcium channels have interesting differences in conduction properties. Ongoing studies using chimeric channel promise to provide novel insight into the structural determinants of Ca2+ permeation through the pore. In addition, most of what we know about the mechanism of permeation comes from study of L-type (C-class) calcium channels. We are pursuing in-depth analysis of permeation in R-type (E-class) channels, which differs in a number of interesting ways that lead to new insights regarding the basis of Ca2+ permation (DeMaria and Yue, 1999 abstract).
Role of calcium channels in heart and nerve: In heart, we are examining the role of calcium channel beta subunits in regulating current in normal and heart-failure states. Using cultured adult heart cells, and viral expression of various calcium-channel beta subunits, we are uncovering tantalizing hints of a major role of beta-subunit regulation in normal and failing cells (see Wei et al., 1998, abstract in press).
Cultured hippocampal autapses are also providing an interesting model of short-term synaptic responsiveness, design characteristics of which are believed to be crucial for the neurocomputational processing and capabilities. We are currently exploring the role of G-protein modulation of calcium channels and calcium-channel inactivation in regulating short-term depression and facilitation of synaptic efficacy during repetitive stimulation.
In connection with the primary culture models of heart and nerve, we are developing adenoviral transfection strategies to permit inducible expression of foreign genes. The goal here is to predict and observe alterred physiology in response to overexpression or knock-out of specific calcium channel genes, thereby providing new insight into the importance of specific channel properties to cellular function.