One strategy for isolating neuronal L-type calcium (Ca(2+)) currents, which typically comprise a minority of the whole cell current in neurons, has been to use pharmacological agents that increase channel activity. This study examines the effects of the benzoyl pyrrole FPL 64176 (FPL) on L-type Ca(2+) currents and compares them to those of the dihydropyridine (+)-202-791. At micromolar concentrations, both agonists increased whole cell current amplitude in PC12 cells. However, FPL also significantly slowed the rate of activation and elicited a longer-lasting slow component of the tail current compared to (+)-202-791. In single channel cell-attached patch recordings, FPL increased open probability, first latency, mean closed time and mean open time more than (+)-202-791, with no difference in unitary conductance. These gating differences suggest that, compared to (+)-202-791, FPL decreases transition rates between open and closed conformations. Where examined, the actions of FPL and (+)-202-791 on whole cell L-type currents in sympathetic neurons appeared similar to those in PC12 cells. In contrast to its effects on L-type current, 10 microM FPL inhibited the majority of the whole cell current in HEK cells expressing a recombinant N-type Ca(2+) channel, raising caution concerning the use of FPL as a selective L-type Ca(2+) channel agonist in neurons.

This thesis details my examination of several mechanisms for modulation of N-type calcium channels in neonatal rat superior cervical ganglion (SCG) neurons. The first part of this work characterizes cross-talk between two distinct mechanisms of modulation: readily-reversible inhibition induced by activation of heterotrimeric G-proteins (termed G-protein-mediated inhibition), and phosphorylation of the channel by protein kinase C (PKC). Data previously presented by other groups suggested that one effect of activating PKC is to prevent G-protein-mediated inhibition. The goal of this project was to confirm this hypothesis by testing functional competition between these two pathways. My findings show that G-protein-mediated inhibition blocks the effects of activating PKC, and that phosphorylation by PKC blocks G-protein-mediated inhibition, confirming that these two mechanisms are mutually exclusive. In addition, I investigated the effect of activating PKC on whole-cell barium currents in the absence of G-protein-mediated inhibition. When endogenous G-proteins were inactivated by dialyzing the cell with GDP-β-S, a guanine nucleotide that prevents activation of the G-protein's α subunit, activation of PKC with phorbol esters was without obvious effect on whole-cell current amplitude, fast and holding potential-dependent inactivation, and voltage-dependent activation, suggesting that PKC's principal role in modulating these currents is to prevent G-protein-mediated inhibition. From these results, I advanced Bean's 1989 model of reluctant and willing gating (induced by G-protein-mediated inhibition and relief of that inhibition, respectively). In this expanded model, reluctant channels, inhibited by G-proteins, are resistant to phosphylation by PKC (reluctant/P-resistant). Unmodulated channels are called willing/available, as they exhibit willing gating, and are available for either binding to a G-protein or phosphorylation by PKC. Finally, phosphorylation of a willing/available channel by PKC drives the channel into the willing/G-resistant state, in which the channel gates willingly, and is resistant to G-protein-mediated inhibition. These results are published in the Journal of General Physiology(2000; 115:277-286), and are presented in this thesis as Chapter II. In addition to membrane-delimited inhibition, N-type calcium channels are also subject to inhibition via a diffusible second-messenger pathway. In SCG neurons, this inhibition can be observed following stimulation of M1 muscarinic receptors by the agonist oxotremorine-M. Our lab previously hypothesized that the diffusible messenger involved might be the polyunsaturated fatty acid arachidonic acid (AA). To test this hypothesis, our lab examined the effect of bath-applied AA on whole-cell SCG calcium currents, and demonstrated that AA induces inhibition with similar properties as M1 muscarinic inhibition. An analysis of AA's effects on unitary N-type calcium currents, published by Liu and Rittenhouse in Journal of Physiology(2000; 525:391-404), revealed that this inhibition is mediated, at least in part, by both a significant increase in the occurrence of null-activity sweeps and a significant decrease in mean closed dwell time. Based on these results, our lab conducted an examination of AA's effects on whole-cell currents in SCG neurons, and found that AA-induced inhibition is mediated by an increase in holding potential-dependent inactivation and appears independent of AA metabolism. When I examined AA's effects in greater detail, I discovered that, in addition to inhibition, AA also appeared to cause significant enhancement of whole-cell currents. The results characterizing AA's general effects on whole-cell calcium currents in SCG neurons have been published in American Journal of Physiology - Cell Physiology(2001; 280:C1293-C1305). Because my finding that AA enhances whole-cell neuronal calcium currents revealed a novel pathway through which this current can be modulated, I proceeded to characterize this effect. My results showed that enhancement develops significantly faster than inhibition, suggesting different mechanisms or pathways. In addition, dialyzing the cell with BSA, a protein that binds fatty acids, blocked the majority of AA-induced inhibition, but did not reduce enhancement, suggesting that enhancement is independent of inhibition and might be mediated at an extracellular site. Using fatty acid analogs that cannot cross the cell membrane, I confirmed that enhancement occurs extracellularly. My data also indicate that AA-induced enhancement of whole-cell currents does not require metabolism of AA, consistent with enhancement being mediated directly by AA. I also examined the biophysical characteristics of enhancement, and found that both an increase in the voltage sensitivity of activation and an increase in activation kinetics underlie this effect. Finally, using both pharmacological agents and a recombinant cell line, I presented the first demonstration that AA enhances N-type calcium current. These findings are described in detail in a paper recently published in American Journal of Physiology - Cell Physiology(2001; 280:C1306-C1318), and are presented in this thesis as Chapter III. In our investigation of AA's effects on whole-cell calcium currents, we utilized a voltage protocol, in conjunction with pharmacology, to enhance the level of L-type current in these cells. Since whole-cell calcium currents in SCG neurons are comprised of mostly (80-85%) N-type current, with the remaining current comprised of mostly L-type current, this approach allowed us to examine both N- and L-type currents. When currents are recorded in the presence of 1 μM FPL 64174 (FPL), a benzoyl pyrrole L-type calcium channel agonist first described in 1989, stepping the membrane potential to -40 mV following a test pulse to +10 mV generates a slowly-deactivating ("tail") current. This tail current is made up entirely of L-type current, and allows us to readily investigate the effect of various modulatory mechanisms on this current type. Although FPL has been used for almost a decade to study L-type calcium currents, activity of FPL on N-type calcium currents has not been investigated. Because our lab routinely uses micromolar concentrations of FPL to measure whole-cell and unitary calcium currents in neuronal cells, I tested whether FPL has any effects on N-type calcium current. Therefore, I examined the effect of FPL on whole-cell calcium currents in an HEK 293 cell line that expresses recombinant N-type calcium channels. Application of 1 and 10 μM FPL caused significant, voltage-independent inhibition of currents, demonstrating that FPL inhibits N-type calcium current. Thus, at micromolar concentrations, FPL is not selective for L-type calcium current, and any examination of its effects on whole-cell calcium currents should take this into account. The results describing FPL's effects on L- and N-type calcium currents are included in a manuscript currently in preparation, and are presented as Chapter IV.

We examined the effects of arachidonic acid (AA) on whole cell Ca(2+) channel activity in rat superior cervical ganglion neurons. Our companion paper (Liu L, Barrett CF, and Rittenhouse AR. Am J Physiol Cell Physiol 280: C1293-C1305, 2001) demonstrates that AA induces several effects, including enhancement of current amplitude at negative voltages, and increased activation kinetics. This study examines the mechanisms underlying these effects. First, enhancement is rapidly reversible by bath application of BSA. Second, enhancement appears to occur extracellularly, since intracellular albumin was without effect on enhancement, and bath-applied arachidonoyl coenzyme A, an amphiphilic AA analog that cannot cross the cell membrane, mimicked enhancement. In addition, enhancement is voltage dependent, in that currents were enhanced to the greatest degree at -10 mV, whereas virtually no enhancement occurred positive of +30 mV. We also demonstrate that AA-induced increases in activation kinetics are correlated with enhancement of current amplitude. An observed increase in the voltage sensitivity may underlie these effects. Finally, the majority of enhancement is mediated through N-type current, thus providing the first demonstration that this current type can be enhanced by AA.