No abstract

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this ''respiratory burst'' in human neutrophils. Although stoichiometric H ؉ efflux occurs during the respiratory burst, efforts to stimulate voltagegated H ؉ channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H ؉ current properties, each of which increases the H ؉ current at any given voltage: (i) a 40-mV negative shift in the H ؉ conductance-voltage (gH-V) relationship; (ii) faster activation [smaller activation time constant ( act)] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant ( tail)] on repolarization; and (iv) a larger maximum H ؉ conductance (gH,max). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of tail with a time course paralleling the inhibition of electron current. However, the amplitudes of H ؉ and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H ؉ channels rather than inducing a new H ؉ channel.respiratory burst ͉ hydrogen ion ͉ pH ͉ proton channel ͉ ion channels N ADPH oxidase is a major bactericidal weapon of neutrophils and other phagocytes (1). On stimulation, the enzyme complex assembles in the membrane and produces superoxide anion, the precursor to several bactericidal reactive oxygen species. NADPH oxidase is electrogenic (2), releasing a proton into the cytoplasm for each superoxide anion released into the phagocytic vacuole or extracellular medium. As a result, pH changes reflecting increased H ϩ permeability are seen during the respiratory burst in phagocytes (2, 3) after stimulation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Voltagegated proton channels, discovered in snail neurons (4), were postulated to mediate the increased H ϩ permeability during the respiratory burst in human neutrophils (2, 5, 6). The importance of H ϩ efflux as a means of charge compensation is emphasized by the inhibition of PMA-stimulated superoxide anion production by Zn 2ϩ or Cd 2ϩ , which presumably act by inhibiting proton current (7,8). However, attempts to demonstrate effects of PMA on H ϩ currents in phagocytes studied in the whole-cell patchclamp configuration have heretofore been unsuccessful. It is therefore unclear whether the g H observed in phagoc...

BSC1, which was originally identified by its sequence similarity to voltage-gated Na(+) channels, encodes a functional voltage-gated cation channel whose properties differ significantly from Na(+) channels. BSC1 has slower kinetics of activation and inactivation than Na(+) channels, it is more selective for Ba(2+) than for Na(+), it is blocked by Cd(2+), and Na(+) currents through BSC1 are blocked by low concentrations of Ca(2+). All of these properties are more similar to voltage-gated Ca(2+) channels than to voltage-gated Na(+) channels. The selectivity for Ba(2+) is partially due to the presence of a glutamate in the pore-forming region of domain III, since replacing that residue with lysine (normally present in voltage-gated Na(+) channels) makes the channel more selective for Na(+). BSC1 appears to be the prototype of a novel family of invertebrate voltage-dependent cation channels with a close structural and evolutionary relationship to voltage-gated Na(+) and Ca(2+) channels.

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance–voltage relation was half maximal at −59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (−14 vs. −39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795–805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with K i = ∼40 μM, and the HERG-selective blocker, E-4031, blocked with K i = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.