M 1 Muscarinic Modulation of N-Type Calcium Channels: A Dissertation

Abstract

The influx of calcium through N-type calcium channels (N-current) affects a myriad of neuronal functions. These include the triggering of synaptic release of neurotransmitter, adjustment of membrane potential and changes in gene transcription. N-channels are highly modulated proteins, so that N-current is attenuated or potentiated in response to environmental changes. In turn, the modulation of N-current has a direct effect on the downstream events, making the N-channel a focal point in neural signaling, and its modulation a mechanism for short term plasticity. The modulation of N-current by M 1 muscarinic receptors (M 1 Rs) is of particular interest for several reasons. The M 1 R is instrumental in both cognition and memory formation as indicated by studies using either pharmacological agents aimed at M 1 Rs or knockout animals lacking M 1 Rs. Clinically, the M 1 R is an important target in the treatment of Alzheimer’s disease. Thus, like the N-channel, the M 1 R is an important element of neural signaling. Moreover, the stimulation of M 1 Rs affects N-current by through signaling pathways which despite being studied for decades, are not completely understood. For my dissertation I have investigated of M 1 R signaling on N-current using electrophysiological recordings of N-current from freshly dissociated neurons and from HEK cells expressing N-channels and M 1 Rs. Asking how one receptor affects one type of calcium channel would seem to be a simple question. However, the answer has many facets. Since M 1 Rs have multiple downstream effects and N-channels are highly modulated proteins, stimulation of M 1 Rs initiates several different pathways which modulate N-current. This thesis aims to unravel some of the complexities of the interactions of two vital components of neuronal signaling. Here I present the results of studies elucidating three different actions of M 1 signaling of N-current modulation. The first study I present here examines the effect of N-channel subunit composition on modulation of N-current. The stimulation of M 1 Rs in superior cervical ganglion (SCG) neurons elicits a distinct pattern of modulation; inhibiting N-current elicited by strong depolarizations and enhancing current elicited by lesser depolarizations. Thus M 1 Rs cause two simultaneous modulatory effects on N-current; increasing voltage sensitivity and decreasing overall conductance. I found the expression of the N-channel’s β subunit (CaVβ) determines the observed effect. Specifically when the isoform CaVβ2a is expressed M 1 stimulation elicits enhancement without inhibition. Conversely, when CaVβ1b, CaVβ3, or CaVβ4 are expressed M 1 stimulation elicits inhibition with out enhancement. These results fit a model in which both the enhancing and inhibiting effects of M 1 stimulation occur in all channels, but typically inhibition dominates. CaVβ2a…