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Biomedical subjects

Nikita Gamper

Publications and source records attributed to Nikita Gamper.

5 recordsLinked to original sources
PubMed2003

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.

Animals↗
PubMed2003

Ceramide inhibits the potassium channel Kv1.3 by the formation of membrane platforms.

Previous studies suggested a central role of sphingomyelin- and cholesterol-enriched membrane rafts in the initiation of signaling via many receptors. Here, we investigated the role of membrane rafts for the function of the voltage-gated potassium channel Kv1.3. We demonstrate that Kv1.3 localizes in the cell membrane to pre-existing small, sphingolipid- and cholesterol-enriched membrane rafts. Transformation of these small rafts to large ceramide-enriched membrane platforms was achieved by stimulation of the endogenous acid sphingomyelinase, addition of exogenous sphingomyelinase or treatment of the cells with C(16)-ceramide and resulted in clustering of Kv1.3 within ceramide-enriched membrane platforms and inhibition of the channel's activity. Likewise, disruption of pre-existing small rafts inhibited Kv1.3 activity. This indicates that intact small membrane rafts are required for Kv1.3 activity and an alteration of the lipid environment of rafts inhibits Kv1.3. These data, thus, may suggest a novel concept for the regulation of ion channels by the cell membrane composition.

Ceramides↗
PubMed2003

Subunit-specific modulation of KCNQ potassium channels by Src tyrosine kinase.

We studied regulation by c-Src tyrosine kinase (Src) of KCNQ1-5 channels heterologously expressed in Chinese hamster ovary (CHO) cells and of native M current in rat sympathetic neurons. Using whole-cell patch clamp, we found that Src modulates currents from KCNQ3, KCNQ4, and KCNQ5 homomultimers, KCNQ2/3 heteromultimers and native M current, but not currents from KCNQ1 or KCNQ2 homomultimers. Src overexpression had two effects: a decrease of current amplitude (4- to 15-fold for cloned channels and approximately 3-fold for M current) and a slowing of activation kinetics by 2-fold. Both Src actions were mostly reversed by bath application of the Src inhibitors erbstatin (20 microm) and PP2 (200 nm), and mimicked by the tyrosine phosphatase inhibitor sodium vanadate (100 microm). Immunoprecipitation and immunoblot analysis showed Src-dependent phosphotyrosine signals associated with KCNQ3, KCNQ4, and KCNQ5 but not with KCNQ1 or KCNQ2 that may be tyrosine phosphorylation of the channel subunits. Expression of a dominant negative Src that cannot phosphorylate substrates had no effect on the current and did not induce phosphotyrosine signals associated with KCNQ3-5 subunits, further indicating that Src actions on KCNQ currents are mediated by tyrosine phosphorylation. Immunostaining and confocal analysis showed no effect of Src overexpression on the abundance of KCNQ3 protein in CHO cells. Finally, experiments using cloned KCNQ2/3 channels, Src and M(1) muscarinic receptors, and sympathetic neurons demonstrated that the actions on KCNQ channels by Src and by muscarinic agonists use distinct mechanisms.

Animals↗
PubMed2002

Identification of cytoplasmic domains within the epithelial Na+ channel reactive at the plasma membrane.

The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600). Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.

Amino Acid Motifs↗