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Novel role of KCNQ2/3 channels in regulating neuronal cell viability.

ABSTRACT: Overactivation of certain K(+) channels can mediate excessive K(+) efflux and intracellular K(+) depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K(+) efflux, intracellular K(+) depletion, and cell death in hippocampal cultures, whereas little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular K(+) concentration. Supporting a mediating role of KCNQ2/3 channels in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in Chinese hamster ovary (CHO) cells initiated caspase-3 activation. Consistently, application of NEM (20 μM, 8 h) in hippocampal cultures similarly caused caspase-3 activation assessed by immunocytochemical staining and western blotting. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondria membrane depolarization, cytochrome c release, formation of apoptosome complex, and apoptosis-inducing factor (AIF) translocation into nuclear. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis.

SUBMITTER: Zhou X

PROVIDER: S-EPMC3017650 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H+ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H+ ions. KCNQ2/3 current was inhibited by H+ ions with an IC50 of 52 nM (pH 7.3) at -60 mV, rising to 2 microM (pH 5.7) at -10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H+ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (Imax) and the voltage dependence of steady-state activation. Reduction of Imax was antagonized by extracellular K+ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of Imax was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state ( approximately 8 pS) to a subconductance state ( approximately 5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H+ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H+ ions was conferred by the KCNQ3 subunit.

Project description:KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

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Novel role of KCNQ2/3 channels in regulating neuronal cell viability.

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