Dual control of cardiac Na⁺–Ca²⁺ exchange by PIP₂: electrophysiological analysis of direct and indirect mechanisms

Abstract

Cardiac Na + -Ca 2+ exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca 2+ is removed or cytoplasmic Na + is increased. Exogenous phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na + . To probe PIP 2 effects in intact cells, we manipulated PIP 2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca 2+ influx) can be strongly inhibited when M1 agonists induce PIP 2 depletion. However, inward currents (i.e. Ca 2+ extrusion) without cytoplasmic Na + can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPγS or the actin cytoskeleton disruptor latrunculin, even in the presence of non-hydrolysable ATP (AMP-PNP). Thus, G-protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton-PIP 2 interactions. Second, to increase PIP 2 we directly perfused PIP 2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP 2 by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KIβ) and a PI4-kinase (PI4KIIα). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIα. This wortmannin-insensitive PI4KIIα was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol-4-phosphate (PIP) and PIP 2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP 2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking.

Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells

“…3C). Such a shift has been observed also in animal cells under similar conditions of increased PtdInsP 2 levels (Yaradanakul et al, 2007; for review, see Suh and Hille, 2008). The direction of this shift is consistent with the NtORK channel protein sensing an increased density of negative surface charges at the internal surface of the plasma membrane, which is consistent, in turn, with the enrichment of the internal leaflet with PtdInsP 2 .…”

Phosphoinositide Kinases Play Key Roles in Norepinephrine- and Angiotensin II-induced Increase in Phosphatidylinositol 4,5-Bisphosphate and Modulation of Cardiac Function

“…These results suggest that PIP 2 in the membrane could come from different pools that are synthesized by different PI4Ks. The type III PI4Ks come into play only when phosphoinositides begin to be depleted in the membrane with continued PLC activation (41). As membrane trafficking to the cell membrane is increased during GqPCR activation, membrane insertion during receptor activation likely brings PI4Ks to the surface membrane, along with other materials required for the synthesis of PIP 2 .…”

Influence of lipids on protein-mediated transmembrane transport

“…NCX1 can be inactivated by decreasing intracellular Ca 2+ or increasing intracellular Na + concentrations (Reeves and Condrescu, 2008). Increased levels of PIP 2 eliminate the inactivation (He et al, 2000;Yaradanakul et al, 2007) and He et al (2000) showed that PIP 2 could bind to the endogenous XIP region following putative TM helix 5 (Iwamoto et al, 1999;Reeves and Condrescu, 2008;Ren et al, 2010). Additional indirect mechanisms of PIP 2 -regulation of NCX1 such as effects on trafficking have also been described (Yaradanakul et al, 2007).…”