INVESTIGATION AND CHARACTERIZATION OF SPLICE VARIATIONS OF L-TYPE Ca2+ CHANNEL, CaV1.3, IN CHICK BASILAR PAPILLA AND RAT COCHLEAR HAIR CELLS: IMPLICATIONS IN HEARING SHEN YIRU MASTER OF SCIENCE, NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 i ACKNOWLEDGMENTS First and foremost, I would like to extend my deepest appreciation and gratitude to my supervisor, Associate Professor Soong Tuck Wah, who was always available to oversee my project and give his expert advice I thank him for his constant guidance and encouragement throughout my PhD training in the lab Thanks also go out to Dejie, Mui Cheng, Tan Fong, Gregory Tan, Ying Ying, Liao Ping and everyone else in the laboratory for their valuable assistance and teaching I am also grateful to our collaborators: Professor David T Yue (electrophysiology of calcium channels), Professor Paul A Fuchs and Dr Hakim Hiel for their expert advice and technical assistance in understanding the physiology of cochlear My sincere thanks also go to my thesis examiners for spending their time on this thesis Lastly, I would like to express my utmost appreciation to my family, for their love and encouragement ii TABLE OF CONTENTS Title page i Acknowledgements ii Table of Contents iii List of publication vi Summary vii List of Tables x List of Figures xi Abbreviations xiii Chapter 1 Introduction 1.1 Voltage-gated calcium channels 1.1.1 L-type voltage-gated calcium channel (LTCCs) 1.1.2 L-type voltage-gated calcium channel subunits structure 1.2 Cav1.3 voltage-gated calcium channels 1.2.1 Alternative splicing of Cav1.3 gene 10 1.2.2 Cav1.3 and cochlea 14 1.2.3 Cav1.3 and other tissues 14 1.3 Mechanism of calcium dependent inactivation (CDI) 16 1.4 The organ of Corti 17 1.5 Anatomy and functional diversity of the cochlea 20 1.6 Objectives of the study 24 Chapter 26 Materials and Methods 26 2.1 27 Tissues preparation and distribution of splice variant, CaV1.3 IQ iii 2.2 Generation of polyclonal antibodies 28 2.3 Protein immunoblotting 29 2.4 Electrophysiological recordings and data analysis 30 2.5 Immunocytochemistry 32 2.5.1 Whole-mount tissues 32 2.5.2 Frozen-sections 33 Chapter 35 Results 35 Part I Alternative splicing at the C-terminus of rat cochlear Cav1.3a1 subunit 36 3.1 Transcript-scanning of Cav1.3a1 subunit of rat cochlear hair cells 36 3.2 Relative abundance of Cav1.3IQD splice variants in developing rat cochlea 42 3.3 Construction of full-length Cav1.3IQD channels 44 3.4 Cav1.3 IQD channels lack Ca2+-dependent inactivation 47 3.5 Elimination of CDI in CaV1.3IQ channels is independent of -subunit isoform 52 3.6 Construction of CaV1.3IQ -GST for protein induction 56 3.7 Characterization of pAb_DIQ and pAb_Cav1.3 specific antibodies 59 3.8 Selective localization of CaV1.3 IQD and CaV1.3IQfull channels within cochlear hair cells 62 Alternative splicing at the I/II loop region of rat cochlear hair cells 66 3.9 Part II Alternative splicing of Cav1.3a1 subunit in chick basilar papilla 70 3.10 Detection of Cav1.3IQD splice variant in whole chick basilar papilla 70 3.11 Detection of Cav1.3IQD splice variant in single cell RT-PCR of chick hair cells 72 3.12 Relative abundance of Cav1.3IQD splice variant in developing chick cochlea 74 3.13 76 Electrophysiological recording of chick cochlear hair cells (THCs) iv 3.14 Selective localization of Cav1.3IQD channels in chick cochlea 78 Part III Alternative splicing of Cav1.3a1 subunit in other tissues 80 3.15 80 Tissue distribution of splice variant Cav1.3 IQD Chapter 84 Discussion and Conclusion 84 4.1 Identification and characterization of splice variant Cav1.3IQD in rat cochlea 85 4.2 Roles of Cav1.3IQD channels in native hair cells 88 4.3 Roles of Cav1.3IQD channels in other tissues 92 4.4 Importance of diminished inactivation of CaV1.3 channels within hair cells 93 Potential mechanisms for switching the inactivation of CaV1.3 channels in hair cells 94 4.6 Tools for in vivo dissection of CaV1.3 function in hair cells 98 4.7 Future plans 98 4.5 References 100 v LIST OF PUBLICATION Yiru Shen, Dejie Yu, Hakim Hiel, Ping Liao, David T Yue, Paul A Fuchs and Tuck Wah Soong Alternative splicing of the CaV1.3 channel IQ domain¾a molecular switch for Ca2+- dependent inactivation within auditory hair cells (Journal of Neuroscience, 26(42): 10690-10699) ABSTRACTS Shen Yiru and Soong Tuck Wah Investigation and characterization of splice variants of L-type Ca channel gene, Cav1.3 in rat cochlear before, during and after onset of hearing (NUS-NUH scientific conference, 2004) Yiru Shen, Dejie Yu, Hakim Hiel, Ping Liao, David T Yue, Paul A Fuchs and Tuck Wah Soong Alternative splicing of the CaV1.3 channel IQ domain¾a molecular switch for Ca2+- dependent inactivation within auditory hair cells (Society of Neuroscience, Atlanta, Georgia 2006) ORAL PRESENTATIONS Shen Yiru Calcium channels in hair cells - 3rd Singapore International Neuroscience Conference From Brain Research to Brain Repair 23-24 May 2006 Yiru Shen Splice variations of L-type Ca 2+ -channel, Cav1.3, in rat cochlear hair cells Department of Neuroscience, Johns Hopkins University School of Medicine 18 Oct 2005 vi SUMMARY L-type Cav1.3 voltage-gated calcium channels play important roles in insulin secretion, regulating pacemaking activities, mediating synaptic neurotransmission in hair cells and learning and memory For some time, a puzzling question asked was about the lack of correlation between the behaviors of the Cav1.3 channels recorded in native hair cells and cloned Cav1.3 channels recorded in heterologous HEK293 cells The native Ca2+ currents flowing through Cav1.3 channels of cochlear hair cells inactivate only little (Zidanic and Fuchs 1995) while those through heterologously expressed Cav1.3 channels in HEK 293 cells so markedly (Xu and Lipscombe 2001) To understand how these Cav1.3 channels are adapted to such unique behavior, as an initial step, we transcript-scanned mRNA obtained from P9 (before onset of hearing) and P28 (after onset of hearing) rat cochlea to determine whether alternative splicing at the C-terminus of Cav1.3 gene may produce a hair cell splice variant that does not inactivate We found that the alternate use of exon 41 acceptor sites generated a splice variant that lost the calmodulin-binding IQ motif in the Cterminus These Cav1.3IQD ( IQ deleted ) channels exhibited a lack of calciumdependent inactivation (CDI) independent of co-expressed b-subunits in HEK293 cells using whole-cell patch recordings Steady-state inactivation (SSI) properties, mainly reflective of voltage-dependent inactivation, were identical for both types of channels (Cav1.3IQD and Cav1.3 IQfull) Hence, CaV1.3 IQD channels not only expressed, but demonstrated selective loss of CDI We confirmed the presence of the identified splice variant, Cav1.3IQD by RT-PCR, Western blot analysis and immunohistochemistry Splice variant specific polyclonal antibodies were raised to determine its distribution profile in the rat cochlear hair cells Cav1.3IQD channel vii immunoreactivity was preferentially localized to the cochlear outer hair cells (OHCs) while that of Cav1.3 IQfull (IQ-possessing) channels labeled the inner hair cells The preferential expression of Cav1.3IQD channels by OHCs suggests that these may play a role in processes other than neurotransmitter release such as electromotility or gene expression Besides analyzing the splicing patterns at the C-terminus of the Cav1.3 gene, we have identified all alternative splicing combinations in the I-II loop region The III loop region in Cav1.3a1 subunit is known to be the location where many patterns of splice variations can be found Detailed analyses of the distribution of intracellular III loop region splice variants revealed tissue specific and developmental regulation Exon 9* was found to be in developmental rat cochlea, heart and brain Interestingly, we find the highest expression of Exon 9* splice variant in the post-natal day rat cochlea (before onset of hearing) It will be of great interest to characterize the physiological role of Cav1.3 channels containing Exon 9* splice variant and determine if any interacting proteins may be isolated or characterized Therefore, this study provides preliminary data to motivate us to look at the expression of the splice variant Cav1.3 IQD channels in other tissues In our recent study, we have found the mossy fiber axons are labelled by the pAb_ IQ antibody (raised against the Cav1.3 IQ splice variant), while the antibodies raised against other regions of the C-terminus (short or long forms) did not label intensely Furthermore, the expression of splice variant Cav1.3 IQ channels have been found in the sinoatrial node (SAN), which suggests that these channels may play important function in cardiac pacemaking It will be of great interest to transcript-scan the entire Cav1.3 gene in the cochlea developmentally for new alternatively spliced exons and more importantly, the construction of full-length Cav1.3 cDNA libraries will enable us to viii determine the actual combinations of the various alternatively spliced exons This study provides essential information of new alternatively spliced exons of Cav1.3 channels which may play diverse roles in the field of hearing sciences ix LIST OF TABLES Table 1: Classification of subunits subunits and effects of co-expression with x References Glowatzki E, Fuchs PA (2002) Transmitter release at the hair cell ribbon synapse Nat Neurosci 5:147-154 Glueckert R, Wietzorrek G, Kammen-Jolly K, Scholtz A, Stephan K, Striessnig J, Schrott-Fischer A (2003) Role of class D L-type Ca2+ channels for cochlear morphology Hear Res 178:95-105 Green GE, Khan KM, Beisel DW, Drescher MJ, Hatfield JS, Drescher DG (1996) Calcium channel subunits in the mouse cochlea J Neurochem 67:37-45 Haeseleer F, Imanishi Y, Maeda T, Possin DE, Maeda A, Lee A, Rieke F, Palczewski K (2004) Essential role of 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Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H (1999) Calmodulin supports both inactivation and facilitation of L-type calcium channels Nature 399:159-162 119 ... whole chick basilar papilla 70 3 .11 Detection of Cav1 .3IQD splice variant in single cell RT-PCR of chick hair cells 72 3 .12 Relative abundance of Cav1 .3IQD splice variant in developing chick cochlea. .. of Cav1 .3IQ splice variants identified at C-terminus of chick basilar papilla 71 Summary of Cav1 .3IQ splice variants in individual hair cells of chick basilar papilla 73 Relative abundance of I-II... I-II loop splice variants in developmental stages of the chick basilar papilla 75 Electrophysiological recording of a single tall hair cell of chick cochlea 77 Figure 3.25 Localization of chick