Dopamine modulation of Ca2+ dependent Cl- current regulates ciliary beat frequency controlling locomotion inTritonia diomedea

Abstract

SUMMARY The physiological mechanisms controlling ciliary beating remain largely unknown. Evidence exists supporting both hormonal control of ciliary beating and control via direct innervation. In the present study we investigated nervous control of cilia based locomotion in the nudibranch mollusc, Tritonia diomedea. Ciliated pedal epithelial (CPE) cells acting as locomotory effectors may be electrically excitable. To explore this possibility we characterized the cells' electrical properties, and found that CPE cells have large voltage dependent whole cell currents with two components. First, there is a fast activating outward Cl- current that is both voltage and Ca2+ influx dependent(ICl(Ca)). ICl(Ca) is sensitive to DIDS and 9-AC, and resembles currents of Ca2+-activated Cl- channels (CaCC). Ca2+ dependence also suggests the presence of voltage-gated Ca2+ channels; however, we were unable to detect these currents. The second current, a voltage dependent proton current(IH), activates very slowly and is sensitive to both Zn2+ and changes in pH. In addition we identify a new cilio-excitatory substance in Tritonia, viz., dopamine. Dopamine, in the 10 μmol l-1-1 mmol l-1 range, significantly increases ciliary beat frequency (CBF). We also found dopamine and Tritonia Pedal Peptide(TPep-NLS) selectively suppress ICl(Ca) in CPE cells,demonstrating a link between CBF excitation and ICl(Ca). It appears that dopamine and TPep-NLS inhibit ICl(Ca) not through changing [Ca2+]in, but directly by an unknown mechanism. Coupling of ICl(Ca) and CBF is further supported by our finding that DIDS and zero [Cl-]outboth increase CBF, mimicking dopamine and TPep-NLS excitation. These results suggest that dopamine and TPep-NLS act to inhibit ICl(Ca),initiating and prolonging Ca2+ influx, and activating CBF excitation.

Nervous control of ciliary beating by Cl-, Ca2+ and calmodulin inTritonia diomedea

“…To confirm the role of Ca 2+ influx for all excitation we used nifedipine to block Ca 2+ in the presence of a number of known CBF stimulants. Zero external Cl -bath solution increased CBF 117.1±17.4%, a significant increase (Fig.·3A) (N=5; Pр0.001) that mimics the increases seen with Cl -channel blockers (Woodward and Willows, 2006). However, the excitatory effect of a Cl --free bath solution is markedly reduced in the presence of 50· mol·l -1 nifedipine (N=5; Pр0.0009) (Fig.·3A), so much so that the residual excitation is indistinguishable from controls (Pр0.10).…”

“…In an accompanying paper (Woodward and Willows, 2006) we present evidence for voltage-gated Ca 2+ channels in CPE cells, and hypothesize that the blockage of Cl -channels by dopamine and TPep-NLS leads to depolarization and increases in CBF. Influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels has also been proposed as a method for increasing [Ca 2+ ] in and CBF (Christopher et al, 1996).…”

“…The link between blockage of Cl -currents (Woodward and Willows, 2006) and Ca 2+ influx is critical. We hypothesize that blockage of Cl -is linked to depolarization.…”

“…In an accompanying paper (Woodward and Willows, 2006), we showed that ciliated pedal epithelial (CPE) cells of Tritonia diomedea possess calcium dependent Cl -currents, and the direct suppression of these currents by dopamine or TPep-NLS leads to an increase in CBF; this result is mimicked by the Clchannel blocker DIDS. However, the mode of action by which Cl -current blockage increases CBF is not understood.…”

“…Ciliary beating may be under hormonal control (Verdugo, 1980;Korngreen and Priel, 1996;Korngreen et al, 1998;Lieb et al, 2002;Barrera et al, 2004) or nervous control via dopamine and neuropeptides (Willows et al, 1997;Woodward and Willows, 2006), serotonin (Goldberg et al, 1994;Christopher et al, 1996;Willows et al, 1997;Nguyen et al, 2001; Doran et al, 2004), acetylcholine (ACh) (Salathe and Bookman, 1999;Zagoory et al, 2001;Zagoory et al, 2002) or depolarization (Aiello and Guideri, 1964;Mackie et al, 1969;Mackie et al, 1976;Murakami and Takahashi, 1975). Work permitting any distinction between these control mechanisms is sparse and this may be due, in part, to technical challenges presented by vertebrate models.…”