My principal research interests are:

• Electrophysiology of nervous and endocrine culture cells;

• Kinetics, pharmacology and modulation of voltage-dependent ion channels, in particular those permeable to calcium

• Dynamics of intracellular calcium studied by fluorescent probes

• Pathways accumulation and mechanisms and toxicity of heavy metals, such as cadmium and lead, in mammalian cells

• Modulation of neurotrasmitter-gated ion channels by metal ions in native and recombinant receptors

The main aim is to elucidate the kinetics, pharmacological and the modulatory mechanisms of calcium channels , both voltage- and ligand-gated. Recently my interest is also focused on heavy metal toxicity, and particularly on the role of ionic channels in heavy metal permeation and the effect of heavy metals on neurotransmitter gated channels.

Techniques:

• patch-recording

• microspectroflourimetry and image analysis

• cell culture and cell biology

• molecular biology

Research activity

• NMDA channels in native and recombinant receptors:
  role of the different subunits in modulation by metal ions

The N-methyl-D-aspartate subtype of glutamate receptor is a very important receptor in the central nervous system, implicated in the mechanisms of synaptic plasticity and high brain functions, as well as in neurological and neurodegenerative diseases. This ligand-gated ionic channel carries the “slow” component of the glutamate-activated postsynaptic current and is endowed with numerous unique properties: it conducts monovalent cations, but has a significantly high calcium permeability, it is tonically blocked by magnesium (Mg²⁺) in a voltage-depedent manner and allosterically modulated by zinc (Zn²⁺), as well as by various other endogenous and exogenous ligands.

Divalent metal ions can affect the NMDA channel activity in a voltage-dependent (Mg-like) or voltage-independent (Zn-like) manner. We have studied the effect of two toxic metals, lead (Pb²⁺) and nickel (Ni²⁺), in native receptor in cerebellar granule neuron from neonatal rats and in recombinant NMDA channel expressed in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells.
Pb²⁺mediates a voltage-independent allosteric modulation in NMDA channels and its inhibition is independent of the subinit present in the recptor (either NR2A or NR2B). On the contrary, Ni²⁺ has a voltage-dependent Mg-like effect, but also causes a voltage-independent inhibition of NR2A-containing receptor activity and a potentiation in NR2B-containing receptor activity. This last effect is similar to polyamine-mediated potentiation.

• Permeation and toxicity of heavy metals in mammalian cells

The susceptibility of the animal tissues to toxic heavy metals, such as cadmium (Cd²⁺) and lead (Pb²⁺), depends on factors that have been started to be identifiedonly recently. We recently studied the mechanisms and the kinetics of these metals uptake in excitable mammalian cells (neurons and endocrine cells) by real time monitoring of metal ion influx with specific fluorescent probes. Both Cd²⁺ and Pb²⁺ permeates the neuronal membrane through the same pathways as Ca2+, but Pb²⁺ is also rapidly taken up through a passive transport system, which still need to be fully elucidated (Usai et al., 1999; Mazzolini et al., 2001).

The role of voltage-dependent calcium channels in Cd²⁺ uptake suggests that cells rich in these membrane protein are more vulnerable to Cd²⁺ poisoning. Indeed, in endocrine cell lines (insulin-secreting HIT-T15 and INS1) a 'pulse' treatment with 30-300 µM Cd for 30 minutes causes apoptosis at later ( at least 16 hours) time, while a similar treatment is completely unharmful on epithelial and connective cell lines (3T3, HeLa cells), which do not express voltage-gated calcium channels. Induction of apoptosis was revealed by genomic DNA fragmentation and DAPI-stained condensed nuclei and can be modulated by the dihydropyridine nimodipine, an antagonist of L-type calcium channels.

(images were recorded 18 hours after wash)

Collaborations

Unitą INFM e Dipartimento di Fisica - Universitą di Genova
Dipartimento di Medicina Sperimentale, Sez. di Farmacologia Universitą di Genova
Dipartimento Ingegneria Biofisica ed Elettronica DIBE - Universitą di Genova
Dipartimento di Fisiologia Cellulare Universitą di Pavia
School of Life Science Jawaharlal Nehru University New Delhi, India
School of Life Sciences, University of Science and Technology of China Hefei, Anhui, China
Department of Biochemistry and Medical Chemistry, Pomeranian Medical University,Szczecin, Poland
University of KwaZulu-Natal, Research Centre for Plant Growth and Development, Scottsville, South Africa