Factors that modulate Ca2+-dependent regulation ofCav2.1 (P/Q-type) Ca2+ channels Open Access

Abstract

Factors that modulate Ca2+-dependent regulation of Ca v 2.1 (P/Q-type) Ca2+ channelsBy Lisa H. Kreiner Activity-dependent changes in the cytoplasmic Ca2+ concentration in neurons initiate a multitude of processes ranging from gene transcription to neurotransmitter release. Voltage-gated Ca2+ channels are important for these processes as they couple membrane depolarization to the influx of Ca2+ ions into the neuron. Since Ca2+ is a second messenger in many cellular pathways, factors that regulate Ca2+ signaling are essential for proper regulation of neuronal function. Ca v 2.1 (P/Q-type) voltage-gated Ca2+ channels undergo a complex feedback regulation mediated by Ca2+ bound calmodulin, which can cause short-term alterations in synaptic efficacy. Ca2+ influx through Ca v 2.1 produces an initial increase (Ca2+-dependent facilitation (CDF)) and gradual decrease (Ca2+-dependent inactivation (CDI)) in Ca2+ current amplitude during high frequency or prolonged stimulation. We investigated two factors that could potentially regulate CDF and/or CDI in neurons, Ca2+ buffering proteins and Ca2+ induced Ca2+ release from intracellular stores. Voltage clamp recordings from either Ca v 2.1 transfected HEK 293T cells or dissociated Purkinje neurons were used to quantify CDF and CDI that occurs in response to depolarization. The effects of the Ca2+ buffering proteins parvalbumin (PV) and calbindin (CB), and the effects of caffeine-induced Ca2+ release from intracellular stores on CDF and CDI were characterized. We found that regulation of CDI by PV and CB is more complex than expected of simple Ca2+ buffers, and absence of these proteins in neurons results in altered Ca v 2.1 subunit expression. Additionally, we found that caffeine, an activator of intracellular Ca2+ release, enhances CDI in transfected cells, suggesting that Ca2+ release from intracellular stores contributes to regulation of Ca v 2.1. Overall, our results suggest that CDI is highly influenced by endogenous factors, which may allow tailoring of Ca2+ regulation to the needs of specific neurons. Manipulation of these factors may prove useful in developing therapeutic strategies to

treat diseases such as migraine, absence epilepsy, spinocerebellar ataxia and other diseases where Ca2+ signaling is altered.

Table of Contents

Chapter1:

Introduction 1

Characterization of voltage-gated Ca2+ currents 1 Ca v 2.1 channels 5

Regulation of Ca v channels by Ca2+ 10 Ca2+ regulation of Ca v 2.1 12

Physiological significance of Ca2+-dependent modulation of Ca v 2.1 14

Regulation of CDI of Ca v 2.1 15

Significance 22

Chapter 2:

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca v 2.1 Ca2+ channels 27

Abstract 27

Introduction 28

Materials and Methods 29

Results 32

Discussion 40

Chapter 3:

Caffeine increases Ca2+-dependent inactivation of Ca v 2.1 (P/Q-type) Ca2+ channels 61

Abstract 61

Introduction 61

Materials and Methods 63

Results 65

Discussion 68

Chapter 4:

Altered Ca2+-feedback to Ca v 2.1 Ca2+ channels in Purkinje neurons lacking parvalbumin and calbindin 79

Abstract 79

Introduction 80

Materials and Methods 81

Results 87

Discussion 94

Chapter 5:

Discussion 116

I Ca and Ca2+ buffering proteins interact to regulate Ca v 2.1 116

Factors that affect feedback regulation of Ca v 2.1 by CICR 120

Potential consequences of altered Ca v 2.1 feedback regulation for neuronal function 123

Role of Ca v 2.1 in network function and pathological conditions 125

References 127

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, Pharmacology
Keyword	Purkinje neuron calbindin parvalbumin calcium channel mouse inactivation
Committee Chair / Thesis Advisor	Lee, Amy, Emory University
Committee Members	Hartzell Jr., Criss, Emory University Finch, Elizabeth, Duke University Traynelis, Stephen, Emory University Abercrombie, Ronald F., Emory University

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