Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the ␣4␤2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different ␣4␤2 subunit ratios (1␣:4␤, 2␣:3␤, and 4␣:1␤) expressed in Xenopus oocytes. The presence of ␣4 subunit in the oocyte plasmatic membrane increased linearly with the amount of ␣4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the ␣4 subunit. The 2␣4:3␤2 subunit ratio displayed the highest ACh sensitivity. Nicotine doseresponse curves for the 1␣4:4␤2 and 2␣4:3␤2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 M. A biphasic curve for 4␣4:1␤2 was obtained at nicotine concentrations higher than 300 M. The 1␣4:4␤2 subunit ratio exhibited the lowest ACh-and nicotine-induced macroscopic current, whereas 4␣4:1␤2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of ␤2:␣4 subunits increased. All three ␣4␤2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2␣4: 3␤2 subunit ratio. Our data suggest that the subunit ratio of ␣4␤2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate ␣4␤2 receptor activation and desensitization. Neuronal nicotinic acetylcholine receptors (nAChRs)1 belong to a superfamily of ligand-gated ion channels (e.g. ␥-aminobutyric acid, glutamate, 5-hydroxytryptamine, among others) and play an important role in modulating neurotransmitter release in distinct areas of the central and peripheral nervous system (1-5). Nicotine is the active ingredient of tobacco and specifically binds to nAChRs in the brain (3). One of the most remarkable effects of chronic nicotine exposure is the up-regulation of the ␣4␤2 subtype in the central nervous system (6 -10). Another important effect of chronic nicotine exposure is the long lasting functional deactivation of nAChR receptor (11-17). Chronic nicotine exposure produces a loss of nicotinic functional activity as a result of rapid and persistent desensitization (11, 18 -20). Desensitization induced by chronic exposure to nicotine has been hypothesized to trigger the up-regulation of the ␣4␤2 nAChR (3, 6, 21-23). The effect of chronic nicotine exposure on the activity of nAChR subtypes may be related to symptoms associated with nicotine addiction (3, 24, 25) such as tolerance, dependence, and withdrawal. In contrast to the aforementioned studies, a recent work suggests that the ␣4␤2 subtype expressed in the stable cell line K-177 functionally up-reg...

The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the ␥M4 transmembrane domain (␥TM4) by introducing tryptophan, at residues 451-462, along the ␥TM4.

The Na+/H+ and K+/H+ exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H+ exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na+/H+ and K+/H+ exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na+/H+ and K+/H+ exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na+/H+ exchange activation increases with cell shrinkage, whereas the rate of Na+/H+ exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K+/H+ exchange activation increases with cell swelling, whereas the rate of K+/H+ exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K+/H+ exchange are reciprocally related to those controlling Na+/H+ exchange.

Ortiz-Acevedo A, Rigor RR, Maldonado HM, Cala PM. Activation of Na ϩ /H ϩ and K ϩ /H ϩ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity.

Recent evidence has demonstrated that arginine vasopressin (AVP) may modulate primary afferent activity of nociceptors in the dorsal horn of the spinal cord. Because nociceptors are group III and IV afferents, spinal AVP also may modulate the activity of group III and IV afferents that cause reflex cardiovascular responses to muscle contraction. Thus, we compared the pressor (mean arterial pressure), myocardial contractile (dP/dt), and heart rate (HR) responses to electrically induced static contraction of the cat hindlimb before and after lumbar intrathecal (IT) injection (L1-L7) of AVP (n = 9), the V1 receptor antagonist d(CH2)5Tyr(Me)AVP (n = 6), the V2 receptor antagonist d(CH2)5[D-Ile2,Ile4,Ala-NH2(9)]AVP (n = 6), and the V2 agonist [Val4,D]AVP (n = 8). After IT injection of AVP (0.1 or 1 nmol) the pressor and contractile responses to static contraction were attenuated by 55 and 44%, respectively. HR was unchanged. Forty-five to 60 min after AVP injection, the contraction-induced pressor and contractile responses were restored to control levels. V1 receptor blockade augmented contraction-induced increases in mean arterial pressure (36%) and dP/dt (49%) but not HR. V2 receptor blockade had no effect on the cardiovascular response to contraction, whereas selective V2 stimulation attenuated the dP/dt (-20%) and HR (-33%) responses but not the pressor response. These results suggest that AVP attenuates the reflex cardiovascular response to contraction by modulating sensory nerve transmission from contracting muscle primarily via a V1 receptor mechanism in the lumbar spinal cord.

We tested the hypothesis that oxytocin (Oxt) acts in the lumbar spinal cord to attenuate reflex pressor (mean arterial pressure, MAP) and heart rate (HR) responses to static hindlimb contraction (i.e., the exercise pressor reflex). Thus we compared MAP and HR responses to electrically stimulated hindlimb static contraction in the anesthetized cat before and after intrathecal injection of Oxt (30 pmol, n = 3; 300 pmol, n = 6; or 3 nmol, n = 6). The 300-pmol dose was most effective; it attenuated the pressor response to static contraction by 39 +/- 10% but had no effect on HR. In three other cats, contraction-induced increases in MAP and HR were monitored before and after intrathecal injection of 300 pmol of Oxt + 300 nmol of the selective Oxt receptor antagonist [d(CH2)5(1),O-Me-Tyr2,Thr4,Tyr9,Orn8]vasotocin. Pretreatment with the antagonist eliminated the effect of Oxt on MAP. In an additional 10 cats, increases in these same variables in response to static contraction were compared before and after intrathecal injection of the Oxt antagonist (30 nmol, n = 3 or 300 nmol, n = 7) into the lumbar spinal cord (L1-L7). Whereas 30 nmol of the Oxt antagonist had no effect, the 300-nmol dose augmented the contraction-induced pressor and HR responses by 28 +/- 7 and 32 +/- 17%, respectively. These data imply that endogenous Oxt modulates the exercise pressor reflex by its action on Oxt receptors in the lumbar spinal cord that can attenuate sensory nerve transmission from skeletal muscle.

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues alphaS287, alphaC412, betaY441, gammaM299, gammaS460, deltaM293, deltaS297 and deltaN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [(125)I]-alpha-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC(50)), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC(50). Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.

Phosphorylation of the nicotinic acetyl-choline receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC50. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC50’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.