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During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus-evoked release of von Willebrand Factor from human umbilical vein endothelial cells. We conclude that DIDS inhibits MMP exocytosis and through this mechanism preserves neuronal membrane integrity during pathological stress.

To study the effect of chronically elevated CO2 on the excitability and function of neurons, we exposed mice to 7.5-8% CO2 for ∼2 wk (starting at 2 days of age) and examined the properties of freshly dissociated hippocampal neurons. Neurons from control mice (CON) and from mice exposed to chronically elevated CO2 had similar resting membrane potentials and input resistances. CO2-exposed neurons, however, had a lower rheobase and a higher Na+ current density (580 ± 73 pA/pF; n = 27 neurons studied) than did CON neurons (280 ± 51 pA/pF, n = 34; P < 0.01). In addition, the conductance-voltage curve was shifted in a more negative direction in CO2-exposed than in CON neurons (midpoint of the curve was -46 ± 3 mV for CO2 exposed and -34 ± 3 mV for CON, P < 0.01), while the steady-state inactivation curve was shifted in a more positive direction in CO2-exposed than in CON neurons (midpoint of the curve was -59 ± 2 mV for CO2 exposed and -68 ± 3 mV for CON, P < 0.01). The time constant for deactivation at -100 mV was much smaller in CO2-exposed than in CON neurons (0.8 ± 0.1 ms for CO2 exposed and 1.9 ± 0.3 ms for CON, P < 0.01). Immunoblotting for Na+ channel proteins (subtypes I, II, and III) was performed on the hippocampus. Our data indicate that Na+ channel subtype I, rather than subtype II or III, was significantly increased (43%, n = 4; P < 0.05) in the hippocampi of CO2-exposed mice. We conclude that in mice exposed to elevated CO2, 1) increased neuronal excitability is due to alterations in Na+ current and Na+ channel characteristics, and 2) the upregulation of Na+ channel subtype I contributes, at least in part, to the increase in Na+ current density. [ABSTRACT FROM AUTHOR]

Focuses on a study which investigated the role of calcium influx via L- and N-type calcium channels in the activation of a transient large-conductance Ca&sup2+;-activated K[sup+] current in mouse neocortical pyramidal neurons. General properties of outward K[sup+] currents; Effects of block of BK channel blockers on the transient outward current; Characterization of the inward calcium currents.

Demonstrates the existence of an important functional interaction between the sodium/hydrogen exchanger isoform 1 (NHE1) protein and the voltage-sensitive sodium channel in mice. Manifestation of neurological diseases; Hippocampal neuron functioning; Mechanisms for increased excitability in mutant NHE1 neurons; Interaction disturbance of specific proteins in neuron membrane.

Ca2+-activated K+ currents and their Ca2+ sources through high-threshold voltage-activated Ca2+ channels were studied using whole-cell patch-clamp recordings from freshly dissociated mouse neocortical pyramidal neurons. In the presence of 4-aminopyridine, depolarizing pulses evoked transient outward currents and several components of sustained currents in a subgroup of cells. The fast transient current and a component of the sustained currents were Ca2+ dependent and sensitive to charybdotoxin and iberiotoxin but not to apamin, suggesting that they were mediated by large-conductance Ca2+-activated K+ (BK) channels. Thus, mouse neocortical neurons contain both inactivating and noninactivating populations of BK channels. Blockade of either L-type Ca2+ channels by nifedipine or N-type Ca2+ channels by omega-conotoxin GVIA reduced the fast transient BK current. These data suggest that the transient BK current is activated by Ca2+ entry through both N- and L-type Ca2+ channels. The physiological role of the fast transient BK current was also examined using current-clamp techniques. Iberiotoxin broadened action potentials (APs), indicating a role of BK current in AP repolarization. Similarly, both the extracellular Ca2+ channel blocker Cd2+ and the intracellular Ca2+ chelator BAPTA blocked the transient component of the outward current and broadened APs in a subgroup of cells. Our results indicate that the outward current in pyramidal mouse neurons is composed of multiple components. A fast transient BK current is activated by Ca2+ entry through high-threshold voltage-activated Ca2+ channels (L- and N-type), and together with other voltage-gated K+ currents, this transient BK current plays a role in AP repolarization.

Mice lacking Na(+)/H(+) exchanger 1 (NHE1) suffer from recurrent seizures and die early postnatally. Although the mechanisms for seizures are not well established, our previous electrophysiological work has shown that neuronal excitability and Na(+) current density are increased in hippocampal CA1 neurons of these mutant mice. However, it is unknown whether this increased density is related to altered expression or functional regulation of Na(+) channels. In this work, we asked three questions: is the increased excitability limited to CA1 neurons, is the increased Na(+) current density related to an increased Na(+) channel expression, and, if so, which Na(+) channel subtype(s) is upregulated? Using neurophysiological, autoradiographic, and immunoblotting techniques, we showed that both CA1 and cortical neurons have an increase in membrane excitability and Na(+) current density; Na(+) channel density is selectively upregulated in the hippocampus and cortex (P < 0.05); and Na(+) channel subtype I is significantly increased in the hippocampus and Na(+) channel subtype II is increased in the cortex. Our results demonstrate that mice lacking NHE1 upregulate their Na(+) channel expression in the hippocampal and cortical regions selectively; this leads to an increase in Na(+) current density and membrane excitability. We speculate that neuronal overexcitability due to Na(+) channel upregulation in the hippocampus and cortex forms the basis of epileptic seizures in NHE1 mutant mice.