RESUMO
Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.
Assuntos
Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Algoritmos , Animais , Estimulação Elétrica , Eletrodos , Cinética , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Modelos Biológicos , Técnicas de Patch-Clamp , Canais de Potássio/química , Conformação Proteica , RatosRESUMO
Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.
Assuntos
Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Algoritmos , Animais , Estimulação Elétrica , Eletrofisiologia , Cinética , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Camundongos , Técnicas de Patch-ClampRESUMO
To test directly whether fibrin(ogen) is a key binding site for apolipoprotein(a) [apo(a)] in vessel walls, apo(a) transgenic mice and fibrinogen knockout mice were crossed to generate fibrin(ogen)-deficient apo(a) transgenic mice and control mice. In the vessel wall of apo(a) transgenic mice, fibrin(ogen) deposition was found to be essentially colocalized with focal apo(a) deposition and fatty-streak type atherosclerotic lesions. Fibrinogen deficiency in apo(a) transgenic mice decreased the average accumulation of apo(a) in vessel walls by 78% and the average lesion (fatty streak type) development by 81%. Fibrinogen deficiency in wild-type mice did not significantly reduce lesion development. Our results suggest that fibrin(ogen) provides one of the major sites to which apo(a) binds to the vessel wall and participates in the generation of atherosclerosis.
Assuntos
Afibrinogenemia/metabolismo , Aorta/metabolismo , Apolipoproteínas/metabolismo , Arteriosclerose/metabolismo , Lipoproteína(a) , Animais , Apolipoproteínas/genética , Apoproteína(a) , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Voltage-dependent ionic currents were recorded from squid giant fiber lobe neurons using the whole-cell patch-clamp technique. When applied to the bathing solution, methadone was found to block IK, I Na and I Ca. Both I Na and I Ca were reduced without apparent change in kinetics and exhibited IC(50)'s of 50-100 and 250-500 mu M, respectively, at +10 mV. In contrast, IK was reduced in a time-dependent manner that is well fit by a simple model of open channel block (K(D)= 32+/- or 2 mu M, +60 mV, 10 degrees Celsius). The mechanism of I(K) block was examined in detail and involves a direct action of methadone, a tertiary amine, on K channels rather than an opioid receptor-mediated pathway. The kinetics of I(K) block resemble those reported for internally applied long chain quaternary ammonium (QA) compounds; and recovery from I(K) block is QA-like in its slow time course and strong dependence on holding potential. A quaternary derivative of methadone (N-methyl-methadone) only reproduced the effects of methadone on I(K) when included in the pipette solution; this compound was without effect when applied externally. I(K) block thus appears to involve diffusion of methadone into the cytoplasm and occlusion of the open K channel at the internal QA blocking site by the protonated form of the drug. This proposed mode of action is supported by the pH and voltage dependence of block as well as by the observation that high external K+ speeds the rate of drug dissociation. In addition, the effect of methadone on I(K) evoked during prolonged (300 ms) depolarizations suggests that methadone block may interfere with endogenous K+ channel inactivation. The effects of temperature, methadone stereoisomers, and the methadone-like drugs propoxyphene and nor-propoxyphene on IK block were examined. Methadone was also found to block I(K) in GH3 cells and in chick myoblasts.
Assuntos
Analgésicos Opioides/farmacologia , Metadona/farmacologia , Neurônios/química , Bloqueadores dos Canais de Potássio , Analgésicos Opioides/química , Animais , Células Cultivadas/química , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Embrião de Galinha , Decapodiformes , Dextropropoxifeno/análogos & derivados , Dextropropoxifeno/farmacologia , Estimulação Elétrica , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Metadona/química , Músculo Esquelético/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Hipófise/citologia , Ratos , Receptores Opioides mu/agonistas , Sensibilidade e Especificidade , Estereoisomerismo , TemperaturaRESUMO
Using high resolution capacitance (Cm) measurement techniques, we mapped the kinetics of exocytosis evoked by brief (5-100 ms) depolarizations that activate voltage-dependent Ca2+ channels in rat adrenal chromaffin cells. After correcting the Cm signal for contributions from Na+ channel-gating charge movements, the initial kinetics of exocytosis were consistent with depolarization-triggered release occurring initially from an immediately releasable pool of only approximately 17 secretory vesicles. In contrast, repetitive application of longer depolarizations evoked release from a distinct, larger readily releasable pool of approximately 170 vesicles. Our results suggest that the secretory response of a chromaffin cell to an action potential is limited by the size of the immediately releasable pool rather than by a fusion mechanism that is slower than that at synapses.
Assuntos
Medula Suprarrenal/metabolismo , Exocitose , Medula Suprarrenal/citologia , Animais , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Degranulação Celular/fisiologia , Técnicas de Cultura , Dibucaína/farmacologia , Cinética , Potenciais da Membrana , RatosRESUMO
Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.