To understand the molecular basis of state-dependent pharmacological blockade of voltage-gated Ca2+ channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels (alpha1C, alpha1A, and alpha1E) expressed from cDNA clones transfected into HEK 293 cells. State-dependent blockade figures importantly in the therapeutically desirable property of use-dependent drug action. Verapamil (a phenylalkylamine) and diltiazem (a benzothiazepine) were imperfectly selective, so differences in the state dependence of inhibition could be compared among the various channels. We found only quantitative differences in pharmacological profile of verapamil: half-maximal inhibitory concentrations spanned a 2-fold range (70 microM for alpha1A, 100 microM for alpha1E, and 110 microM for alpha1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of alpha1C channels; alpha1A and alpha1E channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 microM for alpha1C, 220 microM for alpha1E, and 270 microM for alpha1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from alpha1C and alpha1A and localized the structural determinants for state dependence to repeats III and IV of alpha1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant alpha1C construct by changing three amino acids in IVS6 (Y14901, A1494S, 11497M) that have been implicated as key coordinating sites for avid benzothiazepine binding. Although these mutations increased the half-maximal inhibitory concentration of diltiazem inhibition by approximately 10-fold, the state-dependent nature of inhibition was spared. This result points to the existence of physically distinct elements controlling drug binding and access to the binding site, thereby favoring a "guarded-receptor" rather than a "modulated-receptor" mechanism of drug inhibition.

A computational model of single rabbit sinoatrial (SA) node cells has been revised to fit data on regional variation of rabbit SA node cell oscillation properties. The revised model simulates differences in oscillation frequency, maximum diastolic potential, overshoot potential, and peak upstroke velocity observed in cells from different regions of the node. Dynamic properties of electrically coupled cells, each with different intrinsic oscillation frequency, are studied as a function of coupling conductance. Simulation results demonstrate at least four distinct regimes of behavior as coupling conductance is varied: a) independent oscillation (Gc < 1 pS); b) complex oscillation (1 < or = Gc < 220 pS); c) frequency, but not waveform entrainment (Gc > or = 220 pS); and d) frequency and waveform entrainment (Gc > or = 50 nS). The conductance of single cardiac myocyte gap junction channels is about 50 pS. These simulations therefore show that very few gap junction channels between each cell are required for frequency entrainment. Analyses of large-scale SA node network models implemented on the Connection Machine CM-200 supercomputer indicate that frequency entrainment of large networks is also supported by a small number of gap junction channels between neighboring cells.

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Abstract-Effectsof cell-to-cell coupling conductance on dynamics of sinus node cells are examined. Cell models are biophysically detailed, and are based on the kinetic equations developed by Noble et al. [Neuronal and Cellular Oscillators, edited by J. W. Jacklet. Marcel Deckker, New York (1989).] Resistively coupled cell pairs show five regimes of behavior as a function of coupling conductance: (1) independent oscillation for G, < 1 pS; (2) primarily quasiperiodic oscillation for 1 = G, < 116 pS; (3) windows of periodic behavior which undergo period doubling bifurcation to chaos for 116 6 G, < 212 pS; (4) frequency entrainment for G, 2 212 pS; (5) waveform entrainment for G, 3 50 nS. Thus. only 4-S gap junction channels are required for frequency entrainment. This is shown to also be the case for large networks of sinus cells modeled on the Connection Machine CM-5. A biophysically detailed two-dimensional network model of the cardiac atrium has also been implemented on the CM-5 supercomputer. The model is used to study effects of spatially localized inhibition of the Na-K pump. Na overloading produced by pump inhibition can induce spontaneous, propagating ectopic beats within the network. At a cell-to-cell coupling value yielding a realistic plane wave conduction velocity of 60 ems-' pump inhibition in small regions of the network containing as few as 1000 cells can induce propagating ectopic beats.