Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca_v2.1 (P/Q-Type) Calcium Channels

Abstract

Voltage-gated CaV2.1 channels comprise a pore-forming α1Asubunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENTLoss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the CaV2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the CaV2.1 ubiquitin–proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for CaV2.1, RNF138. CaV2.1 protein stability is dynamically regulated by RNF138 and auxiliary α2δ and β subunits. We provide a proof of concept that protecting the human CaV2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients.

Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels

“…The levels of AnkB and Cav2.1 were enriched in control and AnkB glut+/− synaptosome fractions at P14 and P30, confirming localization to synaptic compartments (Fig. 6c) [18, 39–41]. AnkB levels were lower in AnkB glut+/− synaptosomes than control synaptosomes, whereas Cav2.1 levels were unchanged, suggesting that the Cav2.1 synaptic pool remained unaffected.…”

“…AnkB interacts with Cav2.1, the pore-forming subunit of P/Q type VGCCs, in brain tissues isolated from adult mice [17]. Cav2.1 is detected pre- and post- synaptically, with multiple isoforms (ranging from 160 to 230 kDa, including a 190 kDa dominant isoform) expressed in brain [18, 19]. Cav2.1 plays a role in calcium-triggered neurotransmitter release at the presynaptic membrane, where it interacts with vesicle fusion machinery proteins, such as syntaxin 1A [20–22].…”

CAV2 promotes the growth of renal cell carcinoma through the EGFR/PI3K/Akt pathway

“… 8 Higher expression of CAV2 has been associated with different types of cancer progression including lung, prostate, renal and breast cancer. 9 – 12 In breast tumor, CAV2 expression has been strongly associated with high histological grade 13 and poor prognosis. 14 Conflicting observations have been reported by Sagara et al, who found that CAV2 expression was suppressed in breast cancer tissues compared to normal tissues and that the reduced CAV1 was significantly associated with increasing tumor size.…”

Involvement of Parkin in the ubiquitin proteasome system-mediated degradation of N-type voltage-gated Ca2+ channels

“…However, of all E3 enzymes described only two, RFP2 and RFN138, have been found to associate with the pore-forming α 1 subunits of Ca V channels. The first one has been linked to the L-type Ca V 1.2 channels while the second to the P/Q-type Ca V 2.1 channels [ 8 , 14 ].…”

“…These studies indicate that the number of channels may be regulated by ubiquitination and proteasomal degradation, and also show that this process is carried out by specific enzymes of the Ubiquitin Proteasome System (UPS), as is the case of the E3 enzyme RFP2 that promotes the degradation of the Ca V 1.2 channels through an endoplasmic reticulum-associated mechanism known as ERAD [ 8 ]. It is also known that the UPS enzyme RNF14 is present in the microenvironment of the Ca V 2 channels and may regulate its activity [ 13 ], as well as the E3 ubiquitin ligase RNF138 that in conjunction with the auxiliary Ca V α 2 δ and β subunits dynamically regulates the Ca V 2.1α 1 subunit functional expression [ 14 ]. Also, we have recently shown evidence for the regulation of the Ca V 2.2 channels heterologously expressed in HEK-293 cells by the light chain 1 (LC1) of the microtubule-associated protein 1B (MAP1B), via increased ubiquitination of the channels [ 15 , 16 ].…”