Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α3β4, chick α<3/β2, α4β2, α4β4, α7and α8, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α7 and α8 nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α3α4nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α3α4and α3α2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells

α₅ subunit forms functional α₃β₄α₅ nAChRs in transfected human cells

NAChRs heterologously expressid in human cells after transient transfection with α₃β₄α₅ or α₃β₄ subunit cDNAs exhibited similar sensitivities to antogonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of α₅ α₃ and β₄ subunits. One component was comparable to that obtained in α₃β₄-transfected cells, while the additional component, putatively attributed to an α₃β₄α₅ nAChR population, showed a Hill coeeficient &gt; 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the α₅ subunit participates in the assembly of α₃β₄α₅ nAChRs compelxes in human cells, adding a new member to the family of neuronal nicotinic receptors

alpha(5) subunit forms functional alpha(3)beta(4)alpha(5) nAChRs in transfected human cells

NACHRS heterologously expressed in human cells after transient transfection with alpha(3) beta(4) alpha(5) or alpha(3) beta(4) subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha(5), alpha(3) and beta(4) subunits. One component was comparable to that obtained in alpha(3) beta(4)-transfected cells, while the additional component, putatively attributed to an alpha(3) beta(4) alpha(5) nAChR population, showed a Hill coefficient >2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha(5) subunit participates in the assembly of alpha(3) beta(4) alpha(5) nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors

RACK1 is a functional target of the E1A oncoprotein.

The adenoviral E1A proteins have been implicated in promotion of proliferation and transformation, inhibition of differentiation, induction of apoptosis, regulation of transcription, and suppression of tumor growth. The ability of E1A to override the fundamental controls of host cells is based on its ability to physically interact with several cellular proteins. We recently characterized RACK1 as a new E1A-interacting protein. In this report, we show that the extreme N-terminal region of E1A, spanning from aminoacids 1-36, and the conserved WD regions of RACK1 are responsible for this interaction. We also demonstrate that E1A and RACK1 colocalize at the perinuclear membrane in the cells. Furthermore, we provide evidence that E1A is able to antagonize the inhibitory effects of RACK1 on Src activity. These results suggest that RACK1 signaling pathway may be a functional target of E1A, contributing to E1A oncogenic effect in the host cells

E1A deregulates the centrosome cycle in a ran GTPase-dependent manner

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH2-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH2-terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A. These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability