Abstract
Abstract: :
Purpose: Bestrophin, the protein product of the vitelliform macular dystrophy (VMD2/Best disease) gene, has recently been identified as a Ca2+–activated Cl– channel (Sun et al., PNAS 99, 4008–4013, 2002; Tsunenari et al., J Biol Chem 278, 4114–41125, 2003; Qu et al., J Biol Chem 278, 49563–49572, 2003). Several mutants of human bestrophin 1 (hBest1), an isoform selectively expressed in the retinal pigment epithelium (RPE), are responsible for VMD2 and dominantly inhibit the Cl– conductance associated with wild type hBest1 (Qu et al. 2003; Tsunenari et al., 2003). Although the connection between defective hBest1 function and the accumulation of lipofuscin–like material within and beneath the RPE is unknown, it confers to hBest1 a putative key role in the maintenance of a normal retinal function. Therefore, we searched for physiological stimuli capable of modifying hBest1 (ICl,hB1) as well as mouse bestrophin 2 (ICl,mB2) induced Cl– currents in transfected HEK 293 cells. Methods: Recordings were performed using the whole–cell patch–clamp technique. HEK 293 cells were held at 0 mV holding potential, and –100 to +100 mV ramps (150 mV/s) were applied every 8 s after stepping the cell to –100 mV during 500 ms. Extracellular and pipette solutions contained CsCl to eliminate K+ currents. Pipette solution (303 mOsm/kg) contained either 3 nM (low–Ca) or 600 nM (high–Ca) free Ca2+. Results: We found that increasing extracellular osmolarity by 20% (from 303 to 364 mOsm/kg with mannitol or NaCl) induced a ∼60% reduction of both ICl,hB1 (n=13) and ICl,mB2 (n=8) within 3–5 min. This effect was accompanied by clear cell shrinkage. The relative current inhibition was similar with low– (n=11) or high–Ca (n=10) pipette solution, although the control ICl,hB1 and ICl,mB2 current densities were, respectively, 2.3– (n=26) and 4.0–fold (n=16) larger in high–Ca. The inhibition of ICl,hB1 and ICl,mB2 by cell shrinkage was partially reversible upon return to a hypo–osmotic (274 mOsm/kg) solution, which was accompanied by cell swelling. Cell shrinkage or swelling produced no significant change in background current in non transfected 293 cells (n=7), or in cells transfected with an EGFP expression plasmid (n=8). Cell shrinkage or swelling produced either no significant change in the Cl– current obtained in cells transfected with the human CFTR gene and exposed to 50 µM forskolin and 1 mM IBMX (n=6), indicating some specificity of action. Conclusions: We conclude that bestrophin–induced Cl– currents are strongly sensitive to cell volume, which is likely to play a role in the volume changes that RPE cells must experience during their phagocytic activity.
Keywords: ion channels • electrophysiology: non–clinical • retinal pigment epithelium