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High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate.

ABSTRACT: Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (K(d) 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1-14)-ECFP (enhanced cyan fluorescent protein) or NCS-1-ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase delta1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.

SUBMITTER: O'Callaghan DW

PROVIDER: S-EPMC1276920 | biostudies-other | 2005 Oct

REPOSITORIES: biostudies-other

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Project description:Kv7 K(+)-channel subunits differ in their apparent affinity for PIP(2) and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP(2) affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M(1) receptors by oxo-M leads to PIP(2) depletion through G(q) and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M(1) receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC(50) for current suppression was 0.44 +/- 0.08 microM, and the maximal inhibition (Inhib(max)) was 74 +/- 3% (n = 5-7). When tonic PIP(2) abundance was increased by overexpression of PIP 5-kinase, the EC(50) was shifted threefold to the right (1.2 +/- 0.1 microM), but without a significant change in Inhib(max) (73 +/- 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3(T)) that increases whole-cell currents by 30-fold without any change in apparent PIP(2) affinity. Kv7.3(T) currents had a slightly right-shifted EC(50) as compared with Kv7.2/7.3 heteromers (1.0 +/- 0.8 microM) and a strongly reduced Inhib(max) (39 +/- 3%). In contrast, the dose-response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 +/- 8 nM), and Inhib(max) was slightly increased (81 +/- 6%, n = 3-4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP(2) by coexpressing them with Kv7.3(T) subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3(T)) or higher affinity (Kv7.2 (R463E)/Kv7.3(T)) channels, the EC(50) and Inhib(max) were similar to Kv7.4 or Kv7.3(T) homomers (0.12 +/- 0.08 microM and 79 +/- 6% [n = 3-4] and 0.58 +/- 0.07 microM and 27 +/- 3% [n = 3-4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3(T). The resulting heteromer displayed a very low EC(50) for inhibition (32 +/- 8 nM) and a slightly increased Inhib(max) (83 +/- 3%, n = 3-4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP(2) hydrolysis, PIP(2) binding to Kv7-channel subunits, and K(+) current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP(2) affinities.

Project description:The relationship between receptor-induced membrane phosphatidylinositol-4'5'-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCdelta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCdelta-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCdelta-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 microm (95% confidence limit; 192-381 microm), rising to 693 microm (417-1153 microm) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.

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High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate.

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