Structure and function of KATP-channels in inspiratory neurons of mice
Struktur und Funktion von KATP-Kanälen in inspiratorischen Neuronen der Maus
Mirjam Haller
Doctoral thesis
Date of Examination:
2000-04-27
Date of issue:
2000-12-13
Advisor:
Richter, Diethelm Prof. Dr.
Referee:
Felsch, Wolfgang Prof. Dr.
Referee:
Neher, Erwin Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0006-B5A4-C
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Dissertation
Abstract
English
The respiratory center within the brainstem is one of the most active neuronal networks in the brain that generates rhythmic activity for lifetime. Such extreme stability requires efficient processes for activity-dependent adjustment of neuronal excitability. A regulatory factor securing stability comprises ATP-dependent K+-channels (KATP-channels), which link cell metabolism with electrical activity and continuously adjust the excitability of respiratory neurons during normoxia and hypoxia.Here, KATP-channels are investigated in respiratory neurons in vitro using the rhythmic brainstem slice preparation, which allows simultaneous recordings of respiratory output activity from hypoglossal nerve rootlets, patch-clamp data from respiratory neurons, and microfluorometric measurements. Single channel measurements on rhythmically active inspiratory neurons reveal that KATP-channels are persistently active and are periodically modulated. Parameters that quantitatively describe this behavior are defined and used as criterions to test the channels under conditions affecting the respiratory output (hypoxia, elevation of extracellular [K+]). The data indicate that the periodic modulation of KATP-channels is due to submembrane [ATP]-fluctuations of amplitude 5-40 microM following Na+/K+-pump activity during rhythmic respiratory bursting. Model simulations based on the signal of the fluorometric dye mag-fura-2 give an estimate of approximately 500 microM for ATP-depletion during hypoxia. As revealed by single cell polymerase chain reaction (PCR) analysis of amplified aRNA, KATP-channels of inspiratory neurons are composed of Kir6.2 and SUR1 subunits and thus correspond to the pancreatic beta-cell type. Their gating behavior, however, more closely resembles the channel kinetics described for the smooth muscle cell type as it displays at least three closed states and two open states. Finally, the intrinsic optical signal (IOS), which has previously been utilized as an indirect sensor for neuronal activity changes, is investigated. Evidence is presented that mechanisms other than cell volume changes, e.g. mitochondrial volume changes, contribute to the IOS response specifically after application of KATP-channel drugs.
Keywords: K<SUB>ATP</SUB>-channels; inspiratory neuron; respiration; rhythmic slice preparation; Kir6.2; SUR1; intrinsic optical signal
