Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels.

Liheng Shi,Michael L. Ko,Gladys Y.-P. Ko

doi:10.3389/fncel.2017.00232

Abstract

Modulation of ion channels by extracellular proteins plays critical roles in shaping synaptic plasticity. Retinoschisin (RS1) is an extracellular adhesive protein secreted from photoreceptors and bipolar cells, and it plays an important role during retinal development, as well as in maintaining the stability of retinal layers. RS1 is known to form homologous octamers and interact with molecules on the plasma membrane including phosphatidylserine, sodium-potassium exchanger complex, and L-type voltage-gated calcium channels (LTCCs). However, how this physical interaction between RS1 and ion channels might affect the channel gating properties is unclear. In retinal photoreceptors, two major LTCCs are Cav1.3 (α1D) and Cav1.4 (α1F) with distinct biophysical properties, functions and distributions. Cav1.3 is distributed from the inner segment (IS) to the synaptic terminal and is responsible for calcium influx to the photoreceptors and overall calcium homeostasis. Cav1.4 is only expressed at the synaptic terminal and is responsible for neurotransmitter release. Mutations of the gene encoding Cav1.4 cause X-linked incomplete congenital stationary night blindness type 2 (CSNB2), while null mutations of Cav1.3 cause a mild decrease of retinal light responses in mice. Even though RS1 is known to maintain retinal architecture, in this study, we present that RS1 interacts with both Cav1.3 and Cav1.4 and regulates their activations. RS1 was able to co-immunoprecipitate with Cav1.3 and Cav1.4 from porcine retinas, and it increased the LTCC currents and facilitated voltage-dependent activation in HEK cells co-transfected with RS1 and Cav1.3 or Cav1.4, thus providing evidence of a functional interaction between RS1 and LTCCs. The interaction between RS1 and Cav1.3 did not change the calcium-dependent inactivation of Cav1.3. In mice lacking RS1, the expression of Cav1.3 and Cav1.4 in the retina decreased, while in mice with Cav1.4 deletion, the retinal level of RS1 decreased. These results provide important evidence that RS1 is not only an adhesive protein promoting cell-cell adhesion, it is essential for anchoring other membrane proteins including ion channels and enhancing their function in the retina.

Highlights

Interactions between extracellular proteins and ion channels can modulate channel gating and function
Using a similar strategy to determine the interaction between RS1 and Cav1.4, a full-length human rs1 cDNA was inserted into a pBIND vector that encoded a recombinant protein with GAL4 DNA binding domain as the bait
We found that RS1 did not affect the calcium-induced inactivation (CDI) of Cav1.3-L-type voltage-gated calcium channels (LTCCs) in cells co-transfected with RS1, since there was no statistical difference of the f values between the Cav1.3-LTCCs with or without RS1 (Cav1.3 + β2 + α2δ1: 0.385 ± 0.076; Cav1.3 + β2 + α2δ1 + RS1: 0.402 ± 0.084)

Summary

Introduction

Interactions between extracellular proteins and ion channels can modulate channel gating and function. LTCCs are the major calcium channels in retinal neurons, and calcium influx through these channels is essential for cellular calcium homeostasis and neurotransmitter release from photoreceptors, bipolar cells, horizontal cells and amacrine cells (Barnes and Kelly, 2002; Morgans et al, 2005). Two major LTCCs in retinal photoreceptors are Cav1.3 (α1D) and Cav1.4 (α1F): Cav1.3 is present from the inner segment (IS) to the synaptic terminal and is responsible for calcium homeostasis (Firth et al, 2001; Xu et al, 2002; Morgans et al, 2005; Ko et al, 2007), while Cav1.4 is only expressed at the synaptic terminal and is critical in forming photoreceptor ribbon synapses during retinal development (Liu et al, 2013) and is responsible for neurotransmitter release (Strom et al, 1998; Morgans, 2001; Barnes and Kelly, 2002; Morgans et al, 2005; Jia et al, 2014). Mutations of the gene encoding Cav1.4 cause X-linked incomplete congenital stationary night blindness type 2 (CSNB2) in patients (Bech-Hansen et al, 1998; Strom et al, 1998; Zito et al, 2003; Michalakis et al, 2014), while the null mutation of Cav1.3 in mice causes a mild decrease of retinal light responses (Busquet et al, 2010)

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Conclusion