Antisense depletion of β-subunits fails to affect T-type calcium channels properties in a neuroblastoma cell line

Abstract

Voltage-gated calcium channels can be classified into high voltage activated (HVA) and low voltage activated (LVA or T-type) subtypes. The molecular diversity of HVA channels primarily results from different genes encoding their pore-forming α₁ subunits. These channels share a common structure with an α₁ subunit associated with at least two regulatory subunits (β, α₂–δ). Any of the six α₁-related channels identified to date are regulated in their functional properties through an interaction with the ancillary β-subunit. By contrast, the diversity and the molecular identity of LVA or T-type calcium channels have yet to be defined. Whether LVA channels are modulated by a β-subunit, like HVA channels, is unknown. To address this issue, we have used an antisense strategy to inhibit β-subunit expression in the NG 108-15 neuroblastoma cell line. Differentiated NG 108-15 cells express both LVA and HVA channels. We found that LVA currents were unaffected when cells were incubated with β-antisense, while HVA currents were drastically decreased. Since LVA Ca channel currents in NG 108-15 cells are not regulated by β-subunits, it is reasonable to postulate that the pore-forming subunit(s) of these channels lacks an interaction domain with a β-subunit (AID). This molecular feature, which is common to various T-type channels, indicates further that LVA calcium channels belong to a channel family structurally distant from HVA channels.

Introduction

Voltage-gated calcium channels (VGCCs) are important regulators of intracellular Ca²⁺ concentration. Hence, they control a broad range of cellular functions including membrane excitability, neurotransmitter release, contraction, proliferation, activation of genes and enzymes, or neurite elongation. Electrophysiological exploration on isolated cells using patch-clamp techniques revealed the diverse functional properties of these channels (Stea et al., 1995). Based on their sensitivity to membrane depolarization and pharmacological agents, several types have been identified. High voltage activated (HVA) VGCCs are activated by large depolarizations and consist of at least five types including the L-, N-, P–Q-, and R-type channels. Low voltage activated (LVA) or T-type (for transient and tiny) channels correspond to another group of calcium-permeable voltage-gated channels activated by smaller depolarizations.

The structure of all HVA channels identified to date consists of at least three subunits (α₁, α₂–δ, and β) in a 1–1–1 association. The transmembrane α₁ subunit bears the pore of the channel, the binding sites for calcium antagonists, and ancillary β and α₂–δ subunits. Six genes encoding α₁ subunits have been cloned to date (α_1A, α_1B, α_1C, α_1D, α_1E, and α_1S) and assigned to given Ca current types (Snutch and Reiner, 1992). The structurally related α_1C, α_1D, and α_1S genes were shown to encode various L-type channels. The α_1A and α_1B genes were correlated respectively to P/Q-type and N-type channels. The correspondence between the α_1E gene and a native Ca current has been a matter of debate, but it is suggested that it may notably encode R-type channels (Randall and Tsien, 1997). By contrast, no molecular information is yet available on pore-forming subunits of LVA channels.

Structure–function studies on recombinant HVA channels have clearly established the critical regulatory role of the β-subunit. Four genes encoding a variety of β-subunit isoforms have been identified (β₁, β₂, β₃ and β₄) and each of them behaves as a strong modulator of calcium channel activity. The most striking effect that results from the α₁–β interaction is an enhancement of the current amplitude (see Stea et al., 1995). The presence of a β-subunit also modulates electrophysiological properties of calcium currents, inducing a negative shift of the current–voltage relationship and a modification of the current kinetics (Lacerda et al., 1991, Varadi et al., 1991, Neely et al., 1993). The precise regions of interaction between α₁- and β-subunits have been mapped to the cytoplasmic I–II loop of the α₁ subunit (alpha interacting domain or AID) and a highly conserved region of the β-subunit (beta interacting domain or BID; De Waard et al., 1994, Pragnell et al., 1994). Altogether, it was concluded that the β-subunit can be considered as the main endogenous regulator of HVA calcium channel activity.

Several reports have indicated that T-type channels exhibit tissue-specific properties (Chen and Hess, 1990, Huguenard, 1996) and thereafter that T-type currents are encoded by a group of channels distinct from the HVA channel family. However, it was recently shown that α_1E-related channels can mediate LVA currents in atrial myocytes (Priedras-Renteria et al., 1998). Whether some or all T-type pore-forming subunits are regulated by a β-subunit, as is the case for α_1E channels (Bourinet et al., 1996), needs to be defined. Here we have investigated the consequences of antisense mediated β-subunit inhibition on T-type channels expressed in a neuroblastoma–glioma cell line (NG 108-15). This T-type channel subtype exhibits typical slow kinetics, compared to other T-type isoforms (Chen and Hess, 1990). In agreement with recent reports (Strube et al., 1996, Lambert et al., 1997), we show that inhibition of β-subunit synthesis does not alter the properties of the T-type current in NG 108-15 cells, indicating that T-type channels are not regulated by any of the four Ca channel β-subunits identified to date.