A Novel Human Cl- Channel Family Related to Drosophila flightless Locus

Abstract

Large conductance chloride (maxi-Cl ؊ ) currents have been recorded in some cells, but there is still little information on the molecular nature of the channel underlying this conductance. We report here that tweety, a gene located in Drosophila flightless, has a structure similar to those of known channels and that human homologues of tweety (hTTYH1-3) are novel maxi-Cl ؊ channels. hTTYH3 mRNA was found to be distributed in excitable tissues. The whole cell current of hTTYH3 was large enough to be discriminated from the control but emerged only after treatment with ionomycin. Analysis of pore mutants suggested that positively charged amino acids contributed to anion selectivity. Like a maxi-Cl ؊ channel in situ, the hTTYH3 single channel showed 26-picosiemen linear current voltage, complex kinetics, 4,4-diisothiocyanato-stilbene-2,2-disulfonic acid sensitivity, subconductance, and the permeability order of I ؊ > Br ؊ > Cl ؊ . Similarly, hTTYH2 encoded an ionomycin-induced maxi-Cl ؊ channel, but TTYH1 encoded a Ca 2؉ -independent and swelling-activated maxiCl ؊ channel. Therefore, the hTTYH family encoded maxi-Cl ؊ channels of mammals. Further studies on the hTTYH family should lead to the elucidation of physiological and pathophysiological roles of novel Cl ؊ channel molecules.

Pharmacological properties of a pore induced by raising intracellular Ca2+

“…The pore activated by ATP was also blocked by cytoskeleton antagonists, which suggests that cytoskeleton modification is a common mechanism in the opening of both pores. However, our data do not rule out other possibilities of plasma membrane disruption that allow cytosolic components to escape and reseal preventing the death of wounded cells (52,75).…”

Expression of three distinct families of calcium-activated chloride channel genes in the mouse dorsal root ganglion

“…Experiments during the past two decades have established the existence of at least five distinct families of Cl channels: the ClCs, the aminobutyric acid and glycine receptors, the CLCAs, the Bestrophins, and the Tweety proteins [13,23] . Of them, members of three families: CLCAs, Bestrophins and the Tweety proteins have been suggested to be calcium-activated chloride channels.…”

“…Similarly, there is evidence suggesting that Tweety family members are calcium-activated chloride channels [23] . Expression of hTTYH3 in CHO cells generated a unique Clcurrent activated by an increase in the intracellular Ca 2+ concentration.…”

“…Overexpression of Tthy1 shows that it is a membrane protein [18] . Using constructs for overexpression of Httyh-1, -2 and-3 in CHO and HEK cell lines [23,24] , Suzuki et al suggested that hTTYH1 activation is calcium-independent but that of hTTYH2 and hTTHY3 are strongly calcium-dependent. If the mouse orthologues of these proteins retain the same properties, mTtyh2 and mTtyh3 would be reasonable candidates to underlie Ca 2+ -activated chloride currents in DRG neurons.…”

“…Three genes homologous to the Drosophila Tweety gene have been iden-tified in the human genome [23] . Highly-conserved [GENBANK: NM_001001454] [2] ,Ttyh2 [GENBANK: NM_053273] and Ttyh3 [GENBANK: NM_175274].…”