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Phosphorylation modulates the activity of the ATP-sensitive K+ channel in the ventromedial hypothalamic nucleus
V H Routh1, J J McArdle, B E Levin
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Newark 07104, USA. routhvh@umdnj.edu
Insights
The ventromedial hypothalamic nucleus K-ATP channel is regulated by ATP and phosphorylation. Glucose and glibenclamide inhibit channel activity, while phosphorylation state is a key regulator.
Area of Science:
- Neuroscience
- Molecular Biology
- Physiology
Background:
- The ventromedial hypothalamic nucleus (VMH) plays a crucial role in metabolic regulation.
- ATP-sensitive potassium (K-ATP) channels are important in cellular energy sensing and neuronal excitability.
Purpose of the Study:
- To investigate the regulation of K-ATP channels in VMH neurons.
- To elucidate the roles of ATP, glucose, and phosphorylation in VMN K-ATP channel activity.
Main Methods:
- Utilized cell-attached and inside-out patch-clamp electrophysiology on freshly isolated rat VMN neurons.
- Examined the effects of glucose, ATP, ADP, AMPPNP, glibenclamide, H7, and microcystin on VMN K-ATP channel activity.
Main Results:
- Glucose (10 mM) inhibited VMN K-ATP channel activity by 81%.
- ATP inhibited channels in a concentration-dependent manner (1-10 mM), with inhibition persisting with non-hydrolyzable AMPPNP.
- Glibenclamide, H7, and microcystin demonstrated significant inhibitory and stimulatory effects, respectively, on channel activity.
Conclusions:
- ATP inhibits VMN K-ATP channels, and this effect is independent of phosphorylation.
- Phosphorylation state is a critical regulator of VMN K-ATP channel function.
- These findings provide insights into the neurobiological mechanisms of energy homeostasis in the VMH.
Abstract:
Regulation of the ATP-sensitive K+ (K-ATP) channel was examined in cell-attached and inside-out membrane patches of freshly isolated neurons from the ventromedial hypothalamic nucleus (VMN) of 7-14 day old male Sprague-Dawley rats. When inside-out patches were exposed to symmetrical K+, the reversal potential was -2.85 +/- 1.65 mV, the single channel conductance 46 pS, and the total conductance varied as a multiple of this value. Glucose (10 mM) reversibly inhibited channel activity in cell-attached preparations by 81%. In the presence of 0.1 mM ADP, 10, 5, and 1 mM ATP reversibly inhibited VMN K-ATP channels in inside-out patches by 88, 83, and 60%, respectively. This inhibition was not dependent on phosphorylation since 5 mM AMPPNP, the non-hydrolyzable analog of ATP, reversibly inhibited channel activity by 67%. Relatively high concentrations of glibenclamide (100 microM) also reversibly inhibited VMN K-ATP channel activity in cell attached and inside-out patches by 67 and 79%, respectively. Finally, the non-specific kinase inhibitor H7 (200 microM) decreased channel activity by 53% while the non-specific phosphatase inhibitor microcystin (250 nM) increased channel activity by 218%. These data suggest that while the inhibitory effect of ATP is not phosphorylation dependent, phosphorylation state is an important regulator of the VMN K-ATP channel.