Junda Su

Intrinsic membrane properties of locus coeruleus neurons in Mecp2-null mice

Rett syndrome caused by mutations in methyl-CpG-binding protein 2 (Mecp2) gene shows abnormalities in autonomic functions in which brain stem norepinephrinergic systems play an important role. Here we present systematic comparisons of intrinsic membrane properties of locus coeruleus (LC) neurons between Mecp2(-/Y) and wild-type (WT) mice. Whole cell current clamp was performed in brain slices of 3- to 4-wk-old mice. Mecp2(-/Y) neurons showed stronger inward rectification and had shorter time constant than WT cells. The former was likely due to overexpression of inward rectifier K(+) (K(ir))4.1 channel, and the latter was attributable to the smaller cell surface area. The action potential duration was prolonged in Mecp2(-/Y) cells with an extended rise time. This was associated with a significant reduction in the voltage-activated Na(+) current density. After action potentials, >60% Mecp2(-/Y) neurons displayed fast and medium afterhyperpolarizations (fAHP and mAHP), while nearly 90% WT neurons showed only mAHP. The mAHP amplitude was smaller in Mecp2(-/Y) neurons. The firing frequency was higher in neurons with mAHP, and the frequency variation was greater in cells with both fAHP and mAHP in Mecp2(-/Y) mice. Small but significant differences in spike frequency adaptation and delayed excitation were found in Mecp2(-/Y) neurons. These results indicate that there are several electrophysiological abnormalities in LC neurons of Mecp2(-/Y) mice, which may contribute to the dysfunction of the norepinephrine system in Rett syndrome.

The disruption of central CO2 chemosensitivity in a mouse model of Rett syndrome

People with Rett syndrome (RTT) have breathing instability in addition to other neuropathological manifestations. The breathing disturbances contribute to the high incidence of unexplained death and abnormal brain development. However, the cellular mechanisms underlying the breathing abnormalities remain unclear. To test the hypothesis that the central CO(2) chemoreception in these people is disrupted, we studied the CO(2) chemosensitivity in a mouse model of RTT. The Mecp2-null mice showed a selective loss of their respiratory response to 1-3% CO(2) (mild hypercapnia), whereas they displayed more regular breathing in response to 6-9% CO(2) (severe hypercapnia). The defect was alleviated with the NE uptake blocker desipramine (10 mg·kg(-1)·day(-1) ip, for 5-7 days). Consistent with the in vivo observations, in vitro studies in brain slices indicated that CO(2) chemosensitivity of locus coeruleus (LC) neurons was impaired in Mecp2-null mice. Two major neuronal pH-sensitive Kir currents that resembled homomeric Kir4.1 and heteromeric Ki4.1/Kir5.1 channels were identified in the LC neurons. The screening of Kir channels with real-time PCR indicated the overexpression of Kir4.1 in the LC region of Mecp2-null mice. In a heterologous expression system, an overexpression of Kir4.1 resulted in a reduction in the pH sensitivity of the heteromeric Kir4.1-Kir5.1 channels. Given that Kir4.1 and Kir5.1 subunits are also expressed in brain stem respiration-related areas, the Kir4.1 overexpression may not allow CO(2) to be detected until hypercapnia becomes severe, leading to periodical hyper- and hypoventilation in Mecp2-null mice and, perhaps, in people with RTT as well.

High CO2 chemosensitivity versus wide sensing spectrum: a paradoxical problem and its solutions in cultured brainstem neurons

CO2 central chemoreceptors play an important role in cardiorespiratory control. They are highly sensitive to PCO2 in a broad range. These two sensing properties seem paradoxical as none of the known pH‐sensing molecules can achieve both. Here we show that cultured neuronal networks are likely to solve the sensitivity versus spectrum problem with parallel and serial processes. Studies were performed on dissociated brainstem neurons cultured on microelectrode arrays. Recordings started after a 3 week initial period of culture. A group of neurons were dose‐dependently stimulated by elevated CO2 with a linear response ranging from 20 to 70 Torr. The firing rate of some neurons increased by up to 30% in response to a 1 Torr PCO2 change, indicating that cultured brainstem neuronal networks retain high CO2 sensitivity in a broad range. Inhibition of Kir channels selectively suppressed neuronal responses to hypocapnia and mild hypercapnia. Blockade of TASK channels affected neuronal response to more severe hypercapnia. These were consistent with the pKa values measured for these K+ channels in a heterologous expression system. The CO2 chemosensitivity was reduced but not eliminated by blockade of presynaptic input from serotonin, substance P or glutamate neurons, indicating that both pre and postsynaptic neurons contribute to the CO2 chemosensitivity. These results therefore strongly suggest that the physiological PCO2 range appears to be covered by multiple sensing molecules, and that the high sensitivity may be achieved by cellular mechanisms via synaptic amplification in cultured brainstem neurons.

Pontine norepinephrine defects in Mecp2-null mice involve deficient expression of dopamine β-hydroxylase but not a loss of catecholaminergic neurons

Rett syndrome is a neurodevelopmental disorder caused by Mecp2 gene mutations. In RTT patients and Mecp2-null (Mecp2(-/Y)) mice, norepinephrine (NE) content drops significantly, which may play a role in breathing arrhythmia, sleep disorders and sudden death. However, the underlying mechanisms for the NE defect are not fully understood. The NE defect may result from decreased NE biosynthesis, loss of catecholaminergic neurons or both. Although deficiency in tyrosine hydroxylase (TH) has been demonstrated, it is possible that dopamine beta-hydroxylase (DBH), the critical enzyme converting dopamine to NE, is also affected. To test these possibilities, we studied DBH expressions in pontine catecholaminergic neurons of Mecp2(-/Y) mice identified with breathing abnormalities. In comparison to the wild type, Mecp2(-/Y) mice at 2months of age showed approximately 50% decrease in the expressions of DBH and TH, at both protein and mRNA levels in the locus coeruleus (LC) region. Consistently, DBH and TH immunoreactivity was markedly decreased in LC neurons of Mecp2(-/Y) mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2(-/Y) mice, as almost all TH-positive cells expressed DBH. By counting TH-immunoreactive cells in the LC, we found that the Mecp2(-/Y) mice lost only approximately 5% of the catecholaminergic neurons as compared to wild-type, although their LC volume shrank by approximately 15%. These results strongly suggest that the NE defect in Mecp2(-/Y) mice is likely to result from deficient expression of not only TH but also DBH without significant loss of catecholaminergic neurons in the LC.

Modulation of the Heteromeric Kir4.1-Kir5.1 Channel by Multiple Neurotransmitters via Gαq-coupled Receptors

The heteromeric Kir4.1–Kir5.1 channel is a candidate sensing molecule for central CO2 chemoreception. Since central CO2 chemoreception is subject to neural modulations, we performed studies to test the hypothesis that the Kir4.1–Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, including serotonin (5‐HT), substance‐P (SP), and thyrotropin releasing hormone (TRH). The heteromeric Kir4.1–Kir5.1 channel was strongly inhibited by SP, TRH, and 5‐HT when expressed in Xenopus oocytes, whereas these neurotransmitters had no effect on the homomeric Kir4.1 channel. Such an inhibition was dose‐dependent and relied on specific Gαq‐protein‐coupled receptors and protein kinase C (PKC). No direct interaction of the channel with G‐proteins was found. Channel sensitivity to CO2/pH was not compromised with the inhibition by these neurotransmitters, as the channel remained to be inhibited by acidic pH following an exposure to the neurotransmitters. The firing rate of CO2‐sensitive brainstem neurons cultured in microelectrode arrays was augmented by SP or a 5‐HT2A receptor agonist, which was blocked by PKC inhibitors suggesting that PKC underscores the inhibitory effect of SP and 5‐HT in cultured brainstem neurons as well. Immunostaining showed that both Kir4.1 and Kir5.1 proteins were co‐localized in the cultured brainstem neurons. These results therefore indicate that the heteromeric Kir4.1–Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, suggesting a novel neuromodulatory mechanism for the chemosensitivity of brainstem neurons to elevated PCO2 and acidic pH. J. Cell. Physiol. 214:84–95, 2008.

Subunit-stoichiometric evidence for kir6.2 channel gating, ATP binding, and binding-gating coupling.

ATP-sensitive K+ channels are gated by intracellular ATP, allowing them to couple intermediary metabolism to cellular excitability, whereas the gating mechanism remains unclear. To understand subunit stoichiometry for the ATP-dependent channel gating, we constructed tandem-multimeric Kir6.2 channels by selective disruption of the binding or gating mechanism in certain subunits. Stepwise disruptions of channel gating caused graded losses in ATP sensitivity and increases in basal Popen, with no effect on maximum ATP inhibition. Prevention of ATP binding lowered the ATP sensitivity and maximum inhibition without affecting basal Popen. The ATP-dependent gating required a minimum of two functional subunits. Two adjacent subunits are more favorable for ATP binding than two diagonal ones. Subunits showed negative cooperativity in ATP binding and positive cooperativity in channel gating. Joint disruptions of the binding and gating mechanisms in the same or alternate subunits of a concatemer revealed that both intra- and intersubunit couplings contributed to channel gating, although the binding-gating coupling preferred the intrasubunit to intersubunit configuration within the C terminus. No such preference was found between the C and N termini. These phenomena are well-described with the operational model used widely for ligand-receptor interactions.

Hypercapnia modulates synaptic interaction of cultured brainstem neurons

CO(2) is an important metabolic product whose concentrations are constantly monitored by CO(2) chemoreceptors. However, the high systemic CO(2) sensitivity may not be achieved by the CO(2) chemoreceptors without neuronal network processes. To show modulation of network properties during hypercapnia, we studied brainstem neurons dissociated from embryonic rats (P17-19) in multielectrode arrays (MEA) after initial period (3 weeks) of culture. Spike trains of 33,622 pairs of units were analyzed using peri-event histograms (PEH). The amplitude of peri-central peaks between two CO(2)-stimulated units increased and the peak latency decreased during hypercapnia. Similar enhancement of synaptic strength was observed in those sharing a common input. These phenomena were not seen in CO(2)-unresponsive neurons. The amplitude of peri-central peaks between two CO(2) inhibited units also increased without changing latency. Over 60% CO(2)-stimulated neurons studied received mono-/oligosynaptic inputs from other CO(2)-stimulated cells, whereas only approximately 10% CO(2)-unresponsive neurons had such synaptic inputs. A small number of brainstem neurons showed electrical couplings. The coupling efficiency of CO(2)-stimulated but not CO(2)-unresponsive units was suppressed by approximately 50% with high PCO(2). Inhibitory synaptic projections were also found, which was barely affected by hypercapnia. Consistent with the strengthening of excitatory synaptic connections, CO(2) sensitivity of post-synaptic neurons was significantly higher than presynaptic neurons. The difference was eliminated with blockade of presynaptic input. Based on these indirect assessments of synaptic interaction, our PEH analysis suggests that hypercapnia appears to modulate excitatory synaptic transmissions, especially those between CO(2)-stimulated neurons.

Subunit stoichiometry of the Kir1.1 channel in proton-dependent gating

Kir1.1 channel regulates membrane potential and K+ secretion in renal tubular cells. This channel is gated by intracellular protons, in which a lysine residue (Lys80) plays a critical role. Mutation of the Lys80 to a methionine (K80M) disrupts pH-dependent channel gating. To understand how an individual subunit in a tetrameric channel is involved in pH-dependent channel gating, we performed these studies by introducing K80M-disrupted subunits to tandem tetrameric channels. The pH sensitivity was studied in whole-cell voltage clamp and inside-out patches. Homomeric tetramers of the wild-type (wt) and K80M-disrupted channels showed a pH sensitivity almost identical to that of their monomeric counterparts. In heteromeric tetramers and dimers, pH sensitivity was a function of the number of wt subunits. Recruitment of the first single wt subunit shifts the pKa greatly, whereas additions of any extra wt subunit had smaller effects. Single-channel analysis revealed that the tetrameric channel with two or more wt subunits showed one substate conductance at ∼40% of the full conductance, suggesting that four subunits act as two pairs. However, three and four substates of conductance were seen in the tetrameric wt-3K80M and 4K80M channels. Acidic pH increased long-time closures when there were two or more wt subunits. Disruption of more than two subunits led to flicking activity with appearance of a new opening event and loss of the long period of closures. Interestingly, the channel with two wt subunits at diagonal and adjacent configurations showed the same pH sensitivity, substate conductance, and long-time closure. These results thus suggest that one functional subunit is sufficient to act in the pH-dependent gating of the Kir1.1 channel, the channel sensitivity to pH increases with additional subunits, the full pH sensitivity requires contributions of all four subunits, and two subunits may be coordinated in functional dimers of either trans or cis configuration.

Kir6.2 Channel Gating by Intracellular Protons: Subunit Stoichiometry for Ligand Binding and Channel Gating

The adenosine triphosphate-sensitive K+ (KATP) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the KATP channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of KATP channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels.