Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

Abstract

Voltage-dependent Ca 2ϩ channels (Ca V ) are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1-7) that are classified into three main subfamilies according to their high voltageactivated (HVA) 2 gating (Ca V 1 and Ca V 2) or low voltage-activated gating (Ca V 3). In addition to the transmembrane poreforming Ca V ␣1 subunit, Ca V 1 and Ca V 2 channels arise from the multimerization of three other proteins (7): a cytoplasmic Ca V ␤ subunit, a mostly extracellular Ca V ␣2␦ subunit, and calmodulin constitutively bound to the C terminus of Ca V ␣1 (8 -12).A considerable body of work documents the interaction and modulation of the Ca V ␣1 subunit of Ca V 1 and Ca V 2 channels (13-18) by the auxiliary Ca V ␤. The high affinity Ca V ␣1-Ca V ␤ interaction site on the pore-forming Ca V ␣1 subunit is a conserved 18-residue sequence in the I-II linker called the ␣ interaction domain (AID) (19,20) that has been structurally resolved by high resolution x-ray crystallography (21-23). Structural work showed that the AID forms a ␣-helix that binds to the ␣ binding pocket (ABP) in the Ca V ␤ nucleotide kinase (NK) domain. It has been proposed that the MMQKAL cluster of residues within the latter determines the high affinity nanomolar interaction between the two proteins (24 -29). Numerous mutational analyses of the AID residues have correlated the Ca V ␤-induced biophysical modulation with the high affinity binding of Ca V ␤ to the AID peptide in a variety of Ca V ␣1 isoforms for Ca V 1 and Ca V 2 channels (25, 29 -32).The association of Ca V ␣1 and Ca V ␤ subunits is also critical for proper channel maturation and cell surface expression of Ca V 2.2 (17), Ca V 1.2 (33, 34), and Ca V 2.3 (35). In Ca V 2.2, the I-II linker is presumed to play a role in this process (17,18), and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by Ca V ␤1b and Ca V ␤3 (32). In addition, the Ca V ␤2-induced increase in Ca V 1.2 whole cell currents was abolished with the AID-defective YWI/AAA mutant (29), suggesting that high affinity binding of Ca V ␤ onto AID is required to traffic Ca V ␣1 to the plasma membrane. Nonetheless, the unique character of the high affinity AID-ABP interface in the membrane targeting of Ca V ␣1 has been questioned (27, 36 -40). In particular, it has been suggested that Ca V ␤-mediated plasma membrane targeting could be uncoupled from Ca V ␤-mediated modulation of channel gating (26, 41) with important contributions from other intracellular regions (33, 39,(42)(43)(44).In addition to Ca V ␤, the ancillary subunit Ca V ␣2␦ and the ubiquitous calmodulin (CaM) protein have also been proposed to modulate HVA channel maturation and targeting (9). For instance, co-expression of Ca V ␣2␦ promoted the trafficking of the Ca V ␣1 subunit of Ca V 2.2 in COS-7 cells (45), suggesting that Ca V ␣2...

Cooperative Activation of the T-type CaV3.2 Channel

“…The asymmetrical nature of Ca V 3.2 is perfectly suited to study the nature of the inter-domain (or intersubunit in K V channels) interaction during channel activation. Furthermore, the T-type Ca V 3.2 channel can be functionally expressed in the absence of auxiliary subunits unlike HVA Ca V 1.2 (66) and Ca V 2.3 (39,45), thus minimizing indirect effects caused by modulation of gating by Ca V ␤ and Ca V ␣2␦ subunits. Biophysical properties were characterized for 47 single mutants and 20 pairs of glycine mutants.…”

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

“…Accordingly the multiple mutations used include: 4xNQ, N92Q/N348Q/N594Q/N876Q; 5xNQ, N92Q/ N184Q/N468Q/N876Q/N986Q; 6xNQ, N92Q/N184Q/N348Q/ N594Q/N812Q/N876Q; 7xNQ, N92Q/N184Q/N348Q/N594Q/ N812Q/N876Q/N986Q; 13xNQ, N92Q/N136Q/N184Q/N348Q/ N468Q/N585Q/N594Q/N769Q/N812Q/N876Q/N883Q/N986Q/ N1066Q; 14xNQ, N92Q/N136Q/N184Q/N348Q/N468Q/ N585Q/N594Q/N663Q/N769Q/N812Q/N876Q/N883Q/ N986Q/N1066Q; and 16xNQ, N92Q/N136Q/N184Q/N324Q/ N348Q/N468Q/N475Q/N585Q/N594Q/N663Q/N769Q/ N812Q/N876Q/N883Q/N973Q/N986Q. Protein expression of these constructs was confirmed by Western blotting in total cell lysates as described previously (17,22).…”

“…HEKT cells were transfected at 80 -90% confluence (1 million cells per 35-mm culture dish) with similar amounts of DNA (1:1 ratio or 4:4 g) for pCMV-Ca V 1.2 WT and pCMV-based Ca V ␣2␦1 constructs in 10 l of Lipofectamine 2000 (Life Technologies, Inc.) using a DNA/lipid ratio of 1:2.5 (17,22). The pmCherryCa V ␣2␦1-HA construct was either expressed as pmCherryCa V ␣2␦1-HA WT or as pmCherry-Ca V ␣2␦1-HA NQ mutants.…”

“…Cell Culture and Transfections-HEK293T or HEKT (human embryonic kidney 293 cells stably expressing an SV40 temperature-sensitive T antigen) and HEKT cells stably transfected with Ca V ␤3 were grown in Dulbecco's high glucose minimum essential medium (DMEM-HG) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin at 37°C under a 5% CO 2 atmosphere as described elsewhere (17,22). HEKT cells were transfected at 80 -90% confluence (1 million cells per 35-mm culture dish) with similar amounts of DNA (1:1 ratio or 4:4 g) for pCMV-Ca V 1.2 WT and pCMV-based Ca V ␣2␦1 constructs in 10 l of Lipofectamine 2000 (Life Technologies, Inc.) using a DNA/lipid ratio of 1:2.5 (17,22).…”

“…The full complement of auxiliary subunits is required to produce high voltage-activated Ca V 1.2 channels with the properties of the native channels. Ca V ␤ promotes the cell surface density of Ca V 1.2 channels through a high affinity interaction (17) in part by preventing its degradation by the ubiquitin/proteasome system (18). Co-expression of the Ca V ␣2␦ subunit with Ca V ␤-bound Ca V ␣1 increases peak current density and promotes channel activation at more negative voltages (19 -23).…”