A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.
Bottom Line: Neutralization of four arginine residues eliminated plasma membrane binding.Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane.Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.Show MeSH
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Mentions: To assess whether this polybasic motif is sufficient to induce plasma membrane binding when attached to an otherwise cytoplasmic protein, we fused residues 526–554 to the C terminus of GFP (GFP526–554; Fig. 3, A and C). This construct localized to the plasma membrane (Fig. 3A). It also accumulated in the nucleus of the vast majority of transfected cells. Notably, a distinct feature of lipid-interacting polybasic membrane targeting motifs is their similarity to nuclear localization sequences (22), a property that may also account for the nuclear targeting of GFP526–554. However, nuclear staining did not obscure plasma membrane binding (see also Figs. 4A and 5B), and this was also confirmed in dividing cells in which nuclear staining was completely absent (Fig. 3A, GFP526–554, right panel; see also Fig. 5A). Taken together, these findings clearly demonstrate that positive charges at the C-terminal end of the CaV1.2 I-II linker form a plasma membrane binding motif sufficient for translocating the cytoplasmic I-II linker and fused GFP to the plasma membrane.
Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.