N-type calcium channels, widely distributed throughout the nervous system, have been unambiguously linked to a variety of important physiological processes including synaptic transmission. In secretory cells, like chromaffin cells, N-type Ca channels can trigger catecholamine secretion. The purpose of this grant is to characterize novel N-type Ca channels found in chromaffin cells. Typically, N-type Ca channels found in both peripheral and central neurons inactivate. Channel availability is greatly diminished at depolarized holding potentials and during prolonged depolarizations. Some chromaffin cells, in contrast, express N-type channels that do not inactivate. These channels do not exhibit decreased availability at depolarized holding potentials, nor do they show inactivation during long depolarizations. N-type Ca channels are thought to be composed of a pore forming alpha1B subunit and accessory subunits. In order to understand the molecular details of bovine chromaffin cell inactivation, we have cloned the bovine alpha1B and various accessory subunits. We have managed to recreate native non-inactivating properties in the Xenopus oocyte expression system by co-expressing alpha1B, beta2A and alpha2/delta. Channels made with other beta-subunits, beta1b, beta1c, beta2b and beta3a, show inactivation. We wish to identify regions in the beta-subunits that are critical in determining their inactivation properties. When beta2a is co-expressed with neuronal alpha1b subunits, inactivation although slowed, is still present. We wish to identify new regions in the alpha1b subunit that determine their inactivation properties. We will avail ourselves of the opportunity to extensively characterize single channel behavior of these non-inactivating N-type channels. In the past, this family of channels has been described as exhibiting modal behavior. Channels display periods of greatly diminished activity. We will determine whether these low Po periods are true channel gating states or whether they are induced by channel inactivation. G-protein betagamma subunits inhibit N-type Ca channels in a voltage-dependent manner; large depolarizations are thought to promote unbinding of the G-betagamma subunit from the channel. Unfortunately, it has been difficult to study the Ca channel/G-betagamma interactions, in part due to channel inactivation. Detailed studies of the interaction of these channels with G-betagamma subunits are planned. In some chromaffin cells N-type channels inactivate, while in others they do not. In cells which show inactivation, all the N-type channels inactivate. In cells which show no inactivation, no N-type Ca channels inactivate. Chromaffin cells will be segregated into two groups - those cells expressing inactivating and those cells expressing non-inactivating N-type Ca channels. N-type Ca currents can be observed in isolation in chromaffin cells, using appropriate antagonists. Using a variety of stimulation protocols we will determine what effect N-type Ca channel inactivation has on catecholamine release; these experiments should provide insights into neurotransmitter release at synapses regulated by either inactivating or non-inactivating Ca channels.
