Molecular-biological approach to the regulatory mechanism of ciliary muscle contraction

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In the ciliary muscle, the tonic contraction requires a sustained influx of Ca^<2+> through the cell membrane. Very little has hitherto been known about the route(s) of Ca^<2+> influx in this tissue that lacks voltage-gated Ca^<2+> channels. To identify ion channels as the Ca^<2+> entry pathway we investigated effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. We have also examined the expression of the trp channel gene in this smooth muscle tissue by RT-PCR using several sense and anti-sense constructs for human and murine trp's spanning 100-130 amino acid segments which almost entirely cover the putative pore forming region of each trp. The major results obtained are summarized as follows:a. Experiments were carried out at 30℃ using pipettes filled with K^+-free solution containing 100 mM Cs aspartate, 5 mM-BAPTA ([Ca^<2+>]_i=70 nM) and 200 μM-GTP (pH 7.0). CCh evoked an inward current showing polarity reversal at holding potential … More near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCC_L, and NSCC_S) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li^+, Na^+, Cs^+, Mg^<2+>, Ca^<2+>, Sr^<2+> and Ba^<2+>, estimated by total ion replacement procedures, were 0.9 : 1.0 : 1.5 : 0.2 : 0.3 : 0.4 : 0.5 for NSCC_L and 1.0 : 1.0 : 1.8 : 2.5 : 2.6 : 3.2 : 5.0 for NSCC_S.b. Both NSCC_L and NSCC_S were dose-dependently inhibited by 1-100 μM of La^<3+>, Gd^<3+> and SKF96365, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the phasic component.c. Experiments with in-situ membrane patches using pipettes filled with PSS identified a carbachol-activated channel with very similar γ(31±1 pS) and τ(10±1 ms; n=8).d. Replacement of GTP in the pipette solution with GTPγS gradually caused a spontaneous opening of the channel in the absence of CCh. The response to CCh was irreversibly inhibited (rather than augmented) by bath application of 1 μM-thapsigargin.e. The results of RT-PCR indicated that the smooth muscle of the bovine ciliary body expresses a relatively high levels of trp's very similar to human trp3 and trp6, which are thought to construct non-selective cation channels controlled by G-protein-linked pathways. Less