A B S T R A C T The properties of Na channels of the node of Ranvier are altered by neutral, amine, and quaternary local anesthetic compounds. The kinetics of the Na currents are governed by a composite of voltage-and time-dependent gating processes with voltage-and time-dependent block of channels by drug. Conventional measurements of steady-state sodium inactivation by use of 50-ms prepulses show a large negative voltage shift of the inactivation curve with neutral benzocaine and with some ionizable amines like lidocaine and tetracaine, but no shift is seen with quaternary QX-572. However, when the experiment is done with repetitive application of a prepulse-testpulse waveform, a shift with the quaternary cations (applied internally) is seen as well. 1-min hyperpolarizations of lidocaine-or tetracaine-treated fibers restore two to four times as many channels to the conducting pool as 50-ms hyperpolarizations. Raising the external Ca ++ concentration also has a strong unblocking effect. These manipulations do not relieve block in fibers treated with internal quaternary drugs. The results are interpreted in terms of a single receptor in Na channels for the different drug types. Lipid-soluble drug forms are thought to come and go from the receptor via a hydrophobic region of the membrane, while charged and less lipid-soluble forms pass via a hydrophilic region (the inner channel mouth). The hydrophilic pathway is open only when the gates of the channel are open. Any drug form in the channel increases the probability of closing the inactivation gate which, in effect, is equivalent to a negative shift of the voltage dependence of inactivation.

a B S TRAC T A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2-2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E -EK, and a minimum in the reversal potential or conductance as external K + ions are replaced by an equivalent concentration of TI + ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers fi~r hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969.J. Gen. Physiol. 54:553-575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time.Evidence has accumulated, for the sodium channel and for several types of potassium channels of electrically excitable cells, that ions interact with the channel and with other ions in it while diffusing across the membrane (French and Adelman, 1976). An earlier paper of Hille (1975 b) discussed a model of the sodium channel in which the permeating ion must pass across a sequence of four energy barriers to cross the membrane. Inasmuch as the model assumed that no more than one ion could be in the channel at a time, it was called a oneion pore. In this paper we consider a similar type of linear, multibarrier model for a multi-ion pore where more than one ion may be in a channel at a time, and the ions are not permitted to pass by each other as they move through the channel. These assumptions lead to phenomena commonly referred to as "single-file diffusion" or the "long pore effect" which have been reported in measurements of the passive movement of ions in potassium channels of nerve, muscle, and other cell membranes. Our goal is to show that the major transport properties of potassium channels may be accounted for by this class of multi-ion channel models. For practical reasons, only a qualitative agreement is demonstrated here. An attempt to make more realistic models would need many more J. GEN. PHYSIOL. 9 The Rockefeller University Press .

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Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a minority phospholipid of the inner leaflet of plasma membranes. Many plasma membrane ion channels and ion transporters require PIP 2 to function and can be turned off by signaling pathways that deplete PIP 2 . This review discusses the dependence of ion channels on phosphoinositides and considers possible mechanisms by which PIP 2 and analogues regulate ion channel activity.

The CB1 cannabinoid receptor mediates many of the psychoactive effects of ⌬ 9 THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB 1/CB2 receptors may contribute to the behavioral, vascular, and immunological actions of ⌬ 9 THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (⌬ 9 THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve G q, G12, RhoA, actin, phospholipase C, and calcium release from IP 3R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB 1 and CB2.orphan ͉ pain ͉ CB3 ͉ G protein-coupled receptor C annabis has been used and abused for its therapeutic and psychoactive properties for millennia. The effects of cannabinoid compounds are largely mediated by cannabinoid receptors. CB 1 , cloned in 1990 (1), is widely and highly expressed in the CNS, where it likely mediates the majority of the psychotropic and behavioral effects of cannabinoids. CB 2 is primarily expressed in peripheral tissues (2). Both CB 1 and CB 2 are 7-transmembrane G protein-coupled receptors that engage predominantly the G i/o family of G proteins. However, ample evidence suggests that additional receptors may contribute to the behavioral, vascular, and immunological actions of ⌬ 9 tetrahydrocannabinol (THC) and endogenous cannabinoids (3).It has been suggested that GPR55 is a novel cannabinoid receptor (reviewed in ref. 4). GPR55 is only 13.5% identical to CB 1 and 14.4% identical to CB 2 , and its mRNA is present in the brain and periphery (5-7). A recent study found that a variety of cannabinoid compounds stimulated GTP␥S binding in cells stably expressing GPR55 (6). Here, we report GPR55 activation by THC, JWH015, and anandamide increases intracellular calcium by activating signaling pathways quite distinct from those used by CB 1 and CB 2 . Results Activation of GPR55 by Cannabinoids Increases Intracellular Calcium.We first examined the signaling pathways activated by GPR55 in HEK293 cells transiently expressing human GPR55 (hGPR55). Perfusion with 5 M THC evoked a calcium increase (⌬[Ca 2ϩ ] i ) averaging Ϸ100 nM (n ϭ 7, Fig. 1 A and B). Perfusion with 3 M THC evoked a more modest increase (n ϭ 5, 50 nM; Fig. 1B). The agonist-induced calcium response was present in all cells tested, but because it varied in magnitude and time course, concurrent controls were always conducted. GPR55 was essential for the THC-evoked calcium rise because there was minimal calcium rise in nontransfected HEK293 cells exposed to 5 M THC (n ϭ 6, Fig. 1 A and B). A similar calcium increase was seen in CHO cells stably expressing hGPR55 (data not show...

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

Suppression of M current channels by muscarinic receptors enhances neuronal excitability. Little is known about the molecular mechanism of this inhibition except the requirement for a specific G protein and the involvement of an unidentified diffusible second messenger. We demonstrate here that intracellular ATP is required for recovery of KCNQ2/KCNQ3 current from muscarinic suppression, with an EC(50) of approximately 0.5 mM. Substitution of nonhydrolyzable ATP analogs for ATP slowed or prevented recovery. ADPbetaS but not ADP also prevented the recovery. Receptor-mediated inhibition was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP(2)) was blocked by lipid kinase inhibitors. Lipid phosphorylation by PI 4-kinase is required for recovery from muscarinic modulation of M current.

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.