Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity.

The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice i.v. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7.

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.

Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.

Alterations in outward K(+) currents on removal of external Ca(2+) in human atrial myocytes.

External divalent cations are known to play an important role in the function of voltage-gated ion channels. The purpose of this study was to examine the sensitivity of the voltage-gated K(+) currents of human atrial myocytes to external Ca(2+) ions. Myocytes were isolated by collagenase digestion of atrial appendages taken from patients undergoing coronary artery-bypass surgery. Currents were recorded from single isolated myocytes at 37 degrees C using the whole-cell patch-clamp technique. With 0.5 mM external Ca(2+), voltage pulses positive to -20 mV (holding potential = -60 mV) activated outward currents which very rapidly reached a peak (I(peak)) and subsequently inactivated (tau = 7.5 +/- 0.7 msec at +60 mV) to a sustained level, demonstrating the contribution of both rapidly inactivating transient (I(to1)) and non-inactivating sustained (I(so)) outward currents. The I(to1) component of I(peak), but not I(so), showed voltage-dependent inactivation using 100 msec prepulses (V(1/2) = -35.2 +/- 0.5 mV). The K(+) channel blocker, 4-aminopyridine (4-AP, 2 mM), inhibited I(to1) by approximately 76% and reduced I(so) by approximately 33%. Removal of external Ca(2+) had several effects: (i) I(peak) was reduced in a manner consistent with an approximately 13 mV shift to negative voltages in the voltage-dependent inactivation of I(to1). (ii) I(so) was increased over the entire voltage range and this was associated with an increase in a non-inactivating 4-AP-sensitive current. (iii) In 79% cells (11/14), a slowly inactivating component was revealed such that the time-dependent inactivation was described by a double exponential time course (tau(1) = 7.0 +/- 0.7, tau(2) = 90 +/- 21 msec at +60 mV) with no effect on the fast time constant. Removal of external Ca(2+) was associated with an additional component to the voltage-dependent inactivation of I(peak) and I(so) (V(1/2) = -20.5 +/- 1.5 mV). The slowly inactivating component was seen only in the absence of external Ca(2+) ions and was insensitive to 4-AP (2 mM). Experiments with Cs(+)-rich pipette solutions suggested that the Ca(2+)-sensitive currents were carried predominantly by K(+) ions. External Ca(2+) ions are important to voltage-gated K(+) channel function in human atrial myocytes and removal of external Ca(2+) ions affects I(to1) and 4-AP-sensitive I(so) in distinct ways.

L-type calcium current of isolated rat cardiac myocytes in experimental uraemia.

BACKGROUND: End-stage renal failure is associated with a low-output cardiomyopathy, left ventricular hypertrophy and increased QTc dispersion. Cardiac dysfunction is prevalent in patients at the beginning of dialysis and is an important predictor of mortality. Ca(2+) influx through voltage-gated L-type Ca(2+) channels plays a key role in the excitation-contraction coupling of cardiac myocytes. The purpose of this study was to examine the effect of subtotal nephrectomy (SNx) in the rat on both cardiac L-type Ca(2+) currents and action potential duration. METHODS: Wistar rats underwent two-stage SNx; control rats (C) underwent bilateral renal decapsulation. Animals were sacrificed after 8 weeks, and ventricular myocytes were isolated. SNx rats showed a 2-fold increase in plasma urea and creatinine compared with C rats. Whole-cell patch clamp techniques were used to examine L-type Ca(2+) channel currents in isolated cardiac myocytes at 37 degrees C. In separate experiments, the epicardial monophasic action potentials of isolated perfused whole hearts from C and SNx rats were recorded. RESULTS: The amplitude and current-voltage relationships of the L-type Ca(2+) current were not significantly different in myocytes from C (n=11) and SNx (n=8) rats. However, the rate of inactivation of the Ca(2+) current was increased by approximately 15-25% (P<0. 05) in myocytes from SNx rats. The action potential duration (APD(33)) at the apex of the left ventricle was approximately 20% shorter (P<0.01) in hearts from SNx rats as compared with controls. CONCLUSIONS: Renal failure is associated with rapid inactivation of cardiac ventricular myocyte L-type Ca(2+) currents, which may reduce Ca(2+) influx and contribute to shortening of the action potential duration.