I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

Joel P Baumgart,Artem Kondratskyi,Philippe Lory,Edward Perez-Reyes,Isabelle Bidaud,Iuliia Vitko,Arnold Schwartz

doi:10.1371/journal.pone.0002976

Abstract

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3.

Highlights

All excitable cells express voltage-gated Ca2+ channels, and many such cells express a number of voltage-gated Ca2+ channel subtypes [1]
Rationale for deletions Previous studies on Cav3.2 have shown that the proximal portion of the I–II loop contains a gating brake that restrains channels from opening, while the distal I–II loop contains determinants that regulate channel trafficking to the plasma membrane [3,4]
The three Cav3 subunits have in common a proximal domain of the I–II linker that contributes to the gating properties

Summary

Introduction

All excitable cells express voltage-gated Ca2+ channels, and many such cells express a number of voltage-gated Ca2+ channel subtypes [1]. These channels offer a regulated entrance for extracellular Ca2+ into the cell interior, which can initiate a multitude of signaling cascades as Ca2+ itself functions as a secondmessenger. The influx of positively charged Ca2+ ions can effectively depolarize the plasma membrane, which in turn can activate other voltage-gated ion channels. Little is known about the structural determinants of T-channels that underlie their activation at lower voltages than other voltage-gated channels, or what factors control their trafficking to the plasma membrane. Deletions in the distal I–II loop primarily affected trafficking to the plasma membrane [3], and single nucleotide polymorphisms within this loop in Childhood Absence

Methods

Results

Conclusion