RICERCA ITALIANA     

IONIC CHANNELS ACTIVATED BY MEMBRANE HYPERPOLARIZATION AND REGULATED BY CYCLIC NUCLEOTIDES (HCN CHANNELS)

Scuola Internazionale Superiore di Studi Avanzati di Trieste

Abstract

The present proposal aims at clarifying functional properties and at determining molecular mechanisms of ionic channels gated by membrane hyperpolarization and regulated by cyclic nucleotides (CN). In order to understand the physiological properties of HCN channels and the molecular basis of their functions, it is necessary to propose a highly interdisciplinary approach that in Italy could be made exclusively by joining together laboratories of different scientific areas but sharing the same interests. The present proposal will combine the tools more commonly used for ion channel analysis, i.e. biochemistry, structural and molecular biology, electrophysiology, with more theoretical approaches from Bioinformatics and Computational Physics and Chemistry. Our specific aims in the present proposal are:

1 - to study at a molecular level, the composition in subunits and the conformational changes associated with the opening and closure of HCN channels
2 - to build molecular models of HCN channels, to study the changes associated to the opening and the closing of the channels and the possible ways for the interaction among different HCN isoforms
3 - to study the cellular and subcellular localization of the different isoforms in order to try to understand how they are distributed and how the targeting and turnover processes in the membrane are regulated. At this stage, we will try to establish the nature of possible molecular interactors and their >>>

Principal Investigator

Vincent Aldo TORRE Scuola Internazionale Superiore di Studi Avanzati di TRIESTE

Research Objectives

The present proposal aims at clarifying molecular mechanisms and functional properties of ionic channels activated by hyperpolarization and sensitive to cyclic nucleotides(CN).
Ion channels sensitive to CNs fall into different subfamilies of related proteins. Members of one subfamily, designated CNG channels, require cAMP and cGMP for opening but are largely voltage-independent. Members of a second subfamily, the so-called „pacemaker" channels, are activated by hyperpolarization and by CNs (HCN channels).

HCN channels are present in cardiac as well as in many neuronal tissues (Santoro et al, 1997, 1998; Moosmang et al., 2001) and play a fundamental role in visual signal processing in the retina of mammals (Gargini et al., 1999; Demontis et al., 1999,2002). CNG and HCN channels mediate sensory transduction and processing in vision and olfaction. These channels, although belonging to the same super-family of voltage-gated channels, differ significantly from usual K+, Na+ and Ca2+ channels, by their specific gating and selectivity.
We propose a highly interdisciplinary approach to understanding the physiological properties of HCN channels and the molecular basis of their functions. This proposal will combine the usual tools used for ion channel analysis, i.e. biochemistry, structural and molecular biology, electrophysiology, with more theoretical approaches from bioinformatics, computational physics and chemistry.
Such an multidisciplinary >>>

First Results

-Analysis of channels localization in brain tissue and heart
-Analysis of their subcellular localization and identification of subcellular compartments involved in trafficking
-Analysis of channel turnover
-Analysis of channel insertion in me

Timescale

24 months

National and international background

STATE OF THE ART ON THE RESEARCH ON ION CHANNELS
Ionic channels are membrane proteins playing a fundamental role in cell physiology and in signal transduction and transmission (Hille 1992). Potassium, sodium, calcium and chloride ions are used ingeniously by living systems in the performance of fundamental cellular tasks. Through the action of ion pumps, a large fraction of a cell's metabolic energy is spent establishing transmembrane ion gradients. Ion channels have been extensively investigated from an electrophysiological and biochemical point of view and their amino acid sequence determined by molecular biology. The determination of the three dimensional (3D) structure of the K channel (Doyle et al 1998) from the bacteria Streptomices lividans - i.e. the KcsA channel - has opened a new era, allowing us to understand the relation between structure and function of ionic channels at atomic level.

HCN CHANNELS
The so-called Ih current (Di Francesco 1993) flows through HCN channels, discovered in sinoatrial node cells (Brown et al. 1979) and found in hippocampal pyramidal neurons (Halliwell and Adams 1982) and in photoreceptors (Bader et al 1979). HCN channels underlie the rhythmic electric activity of many neurons and of myocytes in the heart. Besides this pace-making function, HCN channels subserve other functions as well. In many neurons HCN channels co-determine resting potential and thereby play an important role in the integrative >>>