In different smooth muscle preparations calcium-activated chloride currents (I_Cl.Ca) contribute to agonist-evoked depolarizations (Large & Wang, 1996) and are therefore an important excitatory conductance. Whilst I_Cl.Ca has been recorded in many smooth muscle cells there has been no information as to the molecular nature of the underlying channel. The aim of the present study was to investigate whether the putative molecular candidates, mClCa1 and mClCa3, underlie this conductance in various murine smooth muscle preparations.

Voltage-clamp experiments were performed on freshly dispersed smooth muscle cells isolated from the portal veins of adult Balb-C mice. Individual animals were sedated by exposure to isoflurane and then killed by cervical dislocation (approved by the Institutional Animal Care and Use Committee). Isolated cells were prepared from dissected tissue for electrophysiology and RT-PCR, as described previously (Duan et al. 2000; Greenwood et al. 2001).

In freshly dispersed murine portal vein myocytes I_Cl.Ca activated by pipette solutions containing 500 nM or 1 µM free Ca²⁺ had characteristics similar to currents activated by this technique in rabbit vascular myocytes (Greenwood et al. 2001). Thus the I_Cl.Ca exhibited time-dependent activation at depolarized potentials that was followed by rapid deactivation upon repolarization, which could be well fitted by a single exponential (mean τ at -40 mV was 95 ± 12 ms, mean ± S.E.M., n = 5). The reversal potential of I_Cl.Ca was close to the theoretical E_Cl and was shifted to -43 ± 2 and +44 ± 1.5 mV (n = 5) when external Cl^– was replaced by SCN^– and isethionate, respectively. The presence of SCN^– in the bathing solution also slowed the rate of deactivation (τ at -40 mV was 188 ± 22 ms; n = 5). So far the only molecular candidates for Ca²⁺-activated chloride currents are members of the recently cloned ClCa family of anion channels. PCR amplification of reverse-transcribed RNA demonstrated the expression of mClCa1 transcripts in isolated smooth muscle cells prepared from murine portal vein, colon, stomach, jejunum, pulmonary artery, as well as murine atrial and ventricular myocytes. mClCa3 transcripts were not detected in any of these isolated muscle cell preparations. We further determined by real-time quantitative RT-PCR the levels of mClCa1 transcriptional expression, within these same tissues. mClCa1 mRNA expression relative to β-actin (arbitary units) was calculated to be 0.057 ± 0.005 for colon, 0.045 ± 0.035 for stomach, 0.145 ± 0.115 for jejunum, 0.043 ± 0.013 for portal vein, 0.035 ± 0.002 for pulmonary artery, 0.161 ± 0.018 for atrium and 0.059 ± 0.006 for ventricle (mean ± S.E.M., n = 3 each tissue).

The expression of mClCa1 transcripts in these isolated cell preparations indicates that mClCa1 may represent a component of I_Cl.Ca in murine portal vein smooth muscle cells.

Duan, D., Ye, L., Britton, F., Horowitz, B. & Hume, J.R. (2000). Circ Res. 86, 485.

Greenwood, I.A., Ledoux, J. & Leblanc, N. (2001). J. Physiol. 534, 395-408. abstract

Large, W.A. & Wang, Q. (1996). Am. J. Physiol. 271, C435-454.