Edward Leon Barsoumian

Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor-type.

N‐type Ca2+ channel modulation by an endogenous P2Y receptor was investigated by the whole‐cell patch‐clamp method in HEK 293 cells transfected with the functional rabbit N‐type calcium channel. The current responses (ICa(N)) to depolarizing voltage steps were depressed by ATP in a concentration‐dependent manner. Inclusion of either guanosine 5′‐O‐(3‐thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration–response curves of ADP and adenosine 5′‐O‐(2‐thiodiphosphate) indicated a higher potency of these agonists than that of ATP, α,β‐methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N6‐(2‐methylthioethyl)‐2‐(3,3,3‐trifluoropropylthio)‐β,γ‐dichloromethylene‐ATP, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid, 2′deoxy‐N6‐methyladenosine‐3′,5′‐diphosphate or 2‐methylthio AMP. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y1, P2Y4, P2Y6, P2Y11 and P2Y13 receptor subtypes, but not the P2Y2, and P2Y12 subtypes, was detected in HEK 293 cells. Immunocytochemistry confirmed the presence of P2Y1, and to a minor extent that of P2Y4, but not of P2Y2 receptors. Hence, it is tempting to speculate that P2Y13 receptors may inhibit N‐type Ca2+ channels via the βγ subunits of the activated Gi protein.

Molecular cloning and characterization of a human brain ryanodine receptor.

We have cloned and sequenced the cDNA of the human brain ryanodine receptor (RyR3), which is composed of 4866 amino acids and shares characteristic structural features with the rabbit RyR3. Northern blot analysis shows that the human RyR3 mRNA is abundantly expressed in hippocampus, caudate nucleus and amygdala as well as in skeletal muscle. The human RyR3 mRNA is also detected in several cell lines derived from human brain tumors. Functional expression of RyR3 and a chimeric RyR suggests that RyR3 forms a calcium‐release channel with a very low Ca2+ sensitivity.

Stable expression and characterization of human PN1 and PN3 sodium channels.

Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.

The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) ‐type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1–GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+‐permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2‐lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip‐mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor‐mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.