Muscarinic receptor-operated cation channels as calcium entry pathways in bovine ciliary muscle

Abstract

In the ciliary muscle, tonic contraction requires a sustained influx of Ca²⁺ through the cell membrane. However, little has been known about the routes for the Ca²⁺ entry in this tissue that lacks voltage-gated Ca²⁺ channels. Recently we have shown by whole-cell voltage clamp experiments that in bovine ciliary muscle cells (BMCs) there are two types of non-selective cation channels (NSCCs) with widely different unitary conductances (35 pS and 100 fS), which are opened by muscarinic stimulation with carbachol (CCh). Here we examined effects of inhibitors of the NSCCs, La³⁺ and Gd³⁺, on CCh-induced changes of the intracellular Ca²⁺ concentration [Ca²⁺]_i. BMCs cultured for 18–48 hours in a serum-free media were used. The [Ca²⁺]_i was monitored by a Fluo-4 fluorescence method. Application of CCh to the BMCs in normal Krebs solution caused an initial phasic increase in the [Ca²⁺]_i followed by a plateau which was abolished by La³⁺ or Gd³⁺ (10-100 μM) as well as by removal of external Ca²⁺. The CCh-induced elevation of [Ca²⁺]_i was also completely inhibited by 100 nM of atropine or 4-DAMP. These results support the idea that the muscarinic receptor-operated NSCCs serve as entry pathway of Ca²⁺ during the sustained phase of contraction. We also conducted immunofluorescence microscopy of the plasma membrane of BCMs and thereby detected transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated NSCCs. [J Physiol Sci. 2006;56 Suppl:S156]