Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

Abstract

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a KD for Ca 2ϩ of ϳ175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN Ϫ ϾI Ϫ ϾCl Ϫ , and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a sitespecific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a poreforming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.

Sarcolemmal Ca 2+ -entry through L-type Ca 2+ channels controls the profile of Ca 2+ -activated Cl − current in canine ventricular myocytes

“…Bestrophin-3, when expressed in HEK cells, was activated by Ca 2+ with the K d of 175 nmol/L, and blocked by micromolar concentrations of DIDS and niflumic acid [62]. The cellular expression of Bestrophin-3 in murine ventricular cells showed a punctuated pattern which was strong in the sarcolemma [62]. In our images obtained in canine ventricular myocytes the cellular expression of Bestrophin-3 showed striated pattern rather than being punctuated.…”

“…The same group reported the expression of Bestrophin-3 in murine and human heart (although only at mRNA level in the latter) similarly to Bestrophin-1 [62]. Bestrophin-3, when expressed in HEK cells, was activated by Ca 2+ with the K d of 175 nmol/L, and blocked by micromolar concentrations of DIDS and niflumic acid [62]. The cellular expression of Bestrophin-3 in murine ventricular cells showed a punctuated pattern which was strong in the sarcolemma [62].…”

“…Moreover, when Bestrophin-1 was extracted and transfected to HEK cells, the resultant current recapitulated the characteristics of I Cl(Ca) , including Ca 2+ -dependence, anion selectivity and sensitivity to DIDS or niflumic acid [61]. The same group reported the expression of Bestrophin-3 in murine and human heart (although only at mRNA level in the latter) similarly to Bestrophin-1 [62]. Bestrophin-3, when expressed in HEK cells, was activated by Ca 2+ with the K d of 175 nmol/L, and blocked by micromolar concentrations of DIDS and niflumic acid [62].…”

The Functional Role of Chloride Channels in Cardiac Pacemaker Activity

“…The molecular identity of CACCs in the heart remains to be determined. CLCA-1 and bestrophins were initially proposed as candidates for CACCs in cardiac tissues (Hartzell et al, 2005;O'Driscoll et al, 2008). It has been demonstrated that at least three members of the murine Bestrophin family, mBest1, mBest2 and mBest3, are expressed in mouse heart.…”

“…It has been demonstrated that at least three members of the murine Bestrophin family, mBest1, mBest2 and mBest3, are expressed in mouse heart. Whole-cell patch clamp experiments with HEK cells transfected with cardiac mBest1 and mBest3 both elicited a calcium sensitive, time independent Cl -current, suggesting mBest1 and mBest3 may function as pore-forming Cl -channels that are activated by physiological levels of calcium (O'Driscoll et al, 2008). Very recently, independent studies from three laboratories have identified a new gene, TMEM16 (or Ano1), as a candidate for CACCs (Schroeder et al, 2008;Caputo et al, 2008;Yang et al, 2008).…”