Comparison of the properties of CLCA1 generated currents and I(Cl(Ca)) in murine portal vein smooth muscle cells

Abstract

Calcium-activated chloride currents (I(Cl(Ca))) have been recorded in various smooth muscle cells but, to date, there has been no information as to the molecular nature of the channel underlying this conductance. We have characterised native I(Cl(Ca)) in freshly dispersed smooth muscle cells isolated from murine portal vein using whole-cell voltage clamp. I(Cl(Ca)) exhibited time-dependent activation at depolarised potentials and rapid deactivation upon repolarisation. The reversal potential of I(Cl(Ca)) was close to the theoretical equilibrium potential (E(Cl)) and was shifted by replacement of external Cl- by SCN- or isethionate. Dithiothreitol (DTT, 1 mM), a blocker of CLCA1, had no effect on the I(Cl(Ca)) current in myocytes. RT-PCR demonstrated the expression of mCLCA1 transcripts, but not mCLCA3 transcripts, in various murine smooth muscle cells including portal vein, as well as cardiomyocytes, and the levels of mCLCA1 transcriptional expression were quantified by real time quantitative RT-PCR. Stable transfection of HEK293 cells with the cDNA encoding mCLCA1 cloned from murine portal vein smooth muscle yielded a current with notable differences in Ca2+ sensitivity, channel kinetics and modulation by DTT from the native I(Cl(Ca)). However, there was some similarity in the pore properties and these data suggest that mCLCA1 alone does not comprise the Cl- channel in portal vein smooth muscle cells.

Figure 1. Characteristics of I_Cl(Ca) evoked in murine portal vein myocytes

Ai, an example of a family of currents evoked by a pipette solution containing 500 nm Ca²⁺ at voltages between −100 mV and +120 mV (voltage protocol is shown in Aii). Superimposed lines show the exponential fit for the outward relaxation at +120 mV and the corresponding tail current recorded at −60 mV. The time constants (τ) for these fits are included. Dashed line represents the zero current level. Bi and Bii show the mean instantaneous and late current densities respectively evoked by 100 nm (▵), 500 nm (•) and 1 μm Ca²⁺ (○). Each point is the mean of between six and 10 cells with error bars representing s.e.m.

Figure 2. Ionic nature of the current evoked in portal vein myocytes

Ai and Aii show families of currents evoked in external solutions containing NaCl, NaSCN or sodium isethionate by 500 nm Ca²⁺ in the pipette solution. The inset shows the voltage protocol used to determine the voltage dependence of the activated current under different external anionic conditions. B, the mean tail current amplitude evoked by 1 μm Ca²⁺ at different test potentials in the presence of external NaCl (•); NaSCN (▴) and sodium isethionate (○).

Figure 3. Lack of effect of the reducing agent DTT on I_Cl(Ca) in portal vein myocytes

A, the amplitude of the instantaneous current (•) and late current (○) recorded at +70 mV in the absence and presence of 1 mm DTT. Insets show ensembles of currents recorded at potentials between −100 mV and +120 mV in the absence and presence of DTT. B, the mean normalised amplitude of the instantaneous and late currents after 5 min application of 1 mm DTT (n = 4).

Figure 4. mCLCA1 expression in murine isolated muscle cells

A, mCLCA1 expression was determined by RT-PCR on isolated smooth muscle and cardiac cell preparations. RT-PCR products were generated through the use of gene-specific primers for mCLCA1. RT-PCR products were resolved on 4 % agarose gels alongside a 100 bp marker. The mCLCA1 cDNA band is indicated at 105 bp. B, the constitutively expressed β-actin was used as a control gene to test the integrity of the cDNA and as a control for genomic DNA contamination since these primers were designed to span an intron as well as two exons. The β-actin amplicon, indicated at 498 bp, confirmed that RT-PCR products were representative of RNA and not contaminated with genomic DNA (an intron-containing amplicon would be 708 bp). C, quantitative levels of mCLCA1 expression in several murine tissues relative to β-actin. mCLCA1 expression was analysed by real-time quantitative PCR using SYBR Green chemistry on an ABI 5700 genetic detector. The same mCLCA1 primers used for qualitative analysis were used for quantitative RT-PCR. The expression of mCLCA1 gene transcripts were calculated from a standard curve (see Methods) and reported relative to β-actin gene expression within the same tissue (arbitrary units). Results are expressed as means ±s.e.m. (n = 3 for each tissue). NTC = no-template control.

Figure 5. Characteristics of I_Cl(Ca) recorded in HEK293 cells stably transfected with mCLCA1

A, the lack of mCLCA1 PCR products in untransfected HEK293 cells (lane 1) but the presence of mCLCA1 amplicons in stably transfected HEK 293 cells (lane 2). β-actin PCR products were detected in both cell types. Bi and Bii show families of currents evoked by pipette solutions containing 500 nm Ca²⁺ (Bi) or 2 mm Ca²⁺ (Bii) recorded at potentials between −100 mV and +100 mV from HEK293 cells transfected with mCLCA1 cloned from portal vein. Dotted line represents the zero current level. C, the mean current density immediately before the end of the test step, evoked by pipette solutions containing 10 mm BAPTA only (zero Ca²⁺, ♦, n = 5); 500 nm Ca²⁺ (⋄; n = 6); and 2 mm Ca²⁺ (▵, n = 14) in transfected HEK293 cells. The density of currents evoked by 2 mm Ca²⁺ in untransfected cells is represented by ▴ (n = 7). D, mean current-voltage relationship for currents evoked by 2 mm Ca²⁺ in HEK293 cells transfected with mCLCA1 in external solutions containing 126 mmNaCl (▵), 126 mmNaSCN (○) or 126 mm sodium isethionate (▴). Each point is the mean of between five and eight cells with error bars representing the s.e.m.

Figure 6. DTT inhibits I_Cl(Ca) recorded in HEK293 cells stably transfected with mCLCA1

A, amplitude of the current recorded at +100 mV in the absence and presence of 1 mm DTT. Inserts show ensembles of currents recorded at potentials between −100 mV and +120 mV in the absence (at the point denoted by *) and presence of DTT (denoted by **). B, mean normalised amplitude of currents recorded at −100 mV () and +100 mV () after 5 min application of 1 mm DTT (n = 4).