The I–II Loop Controls Plasma Membrane Expression and Gating of Ca_v3.2 T-Type Ca²⁺Channels: A Paradigm for Childhood Absence Epilepsy Mutations

Abstract

Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca v 3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca v 3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca V 3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca V 3.2 channel function and expression.

Characterization of the Gating Brake in the I-II Loop of Cav3.2 T-type Ca2+ Channels

“…4 A similar result was reported in a functional study of single nucleotide polymorphisms present in childhood absence epilepsy patients (16), where it was observed that the mutation C456S in Ca v 3.2 channels (located in the proximal I-II loop) shifted the voltage dependence of activation to more negative potentials (17). This observation was followed up with a set of deletions exploring the role of the I-II loop in Ca v 3.2 channels (18). This study revealed that the I-II loop has two separable roles: one to regulate surface expression and another to modulate the biophysical properties of Ca v 3.2 channels.…”

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

“…The ability of zinc to decrease gating charge 25% is consistent with immobilization of one of the four voltage-sensor paddles. It is interesting to note that repeat I plays a dominant role in the opening of both HVA and LVA channels (37,38). These studies also provide evidence for considerable structural similarity between voltage-gated K ϩ and Ca 2ϩ channels, and combined with the established role of Ca v 3.2 in pain and epilepsy, provide a structural model for the development of novel therapeutics (17,24,39,40).…”

The Role of Calcium Channels in Epilepsy

“…When the phenotypic spectrum was extended to include JAE, JME, and temporal lobe epilepsies, more new variants in CACNA1H were identified in affected individuals. When expressed, several of these variants altered T-type channel properties that would be predicted to augment calcium current (Khosravani et al 2005;Peloquin et al 2006;Heron et al 2007) and increase surface expression (Vitko et al 2007). More recently, when the C456S variant was expressed in neurons, it increased neuronal firing, lowered the threshold for rebound burst firing, and induced changes in gene expression (Eckle et al 2014).…”