Paramecium

Paramecium is an excitable unicell.  It responds to various stimuli by reversing its ciliary beat and swim backward for a short distance.  The avoiding reaction is due to a Ca²⁺ action potential that can be readily registered with an electrode.  Over the years, we have selected many behavioral mutants defective in this membrane excitation and cloned some of the corresponding genes. We have then devised a method to clone Paramecium genes by complementing mutant phenotypes (1).  Mutant paramecia unable to generate action potentials and therefore unable to swim backward are called pawns.  The clone pawn gene replenishes the missing Ca²⁺ current in the mutant (1,2) (Fig. 1).

Fig. 1. Ca2+-current phenotypes of Paramecium.  PawnB mutation erases the action potential and therefore the avoiding reaction of Paramecium (1). A. Under a two-electrode voltage clamp, a step depolarization (top trace) induces the Ca2+ action current in the wild type but not the mutant. The pawnB gene was cloned (2). The wild-type transgene in a plasmid restores the Ca2+ action current (bottom trace) in a pawnB transformant cell.  B. Analyses show that the restored current (open square) is quantitatively identical to the wild-type current (filled square) and the untransformed pawnB mutant has no action current (filled squares).

Pantophobiac (pnt) mutants are overly reactive and have a deficit in their Ca²⁺-pendent K⁺ currents.  Fast-2  (fna) mutants are under-reactive and have a deficit in their Ca²⁺-dependent Na⁺ current.  Unexpectedly, both pnt and fna map to the only calmodulin gene in Paramecium.  Further, all fna mutations mapped to the N-terminal lobe and all pnt mapped to the C-lobe of this universal Ca²⁺-binding protein (3,4) (Fig. 2).

Fig. 2  Forward genetics identified a functional bipartition of calmodulin.  Five mutants selected for their “fast-2” phenotype have mutations in the N-terminal lobe of calmodulin.  Seven mutants with the “pantophobaic” phenotype have mutations in the C-lobe.  Electrophysiological examinations showed that each mutant type shows a specific deficit in one kind of Ca2+-CaM dependent channel current.  Amino-acid substitutions of the mutations are marked.

Patch-clamp experiments showed that calmodulin is in fact a detachable subunit of these ion channels (5)  (Fig. 3)

Fig. 3. (Top)  The bipartite distribution of calmodulin (CaM) mutations discovered in vivo from Paramecium.   Ca2+-CaM-dependent Na+ channel and Ca2+-CaM-dependent K+ channel participate in Paramecium membrane excitation.  All mutants showing abbreviated excitation and Na+-current deficit (“fast-2”) are found to have mutations in the N-terminal lobe of calmodulin.  All those with prolonged excitation and K+-current deficit (“pantophobiac”) have mutations in the C-terminal lobe.  The results indicate lobe-specific interactions between calmodulin and different ion channels.  (Bottom) Direct activation of the Paramecium Na+ channels by Ca2+-calmodulin.  Left shows a diagram of channel activation -inactivation cycle.  Right shows unitary activities of Na+ channels at stages corresponding to the diagram.  After inactivation (B), Ca2+ alone cannot restore channel activities (C).  The addition of both Ca2+ and CaM are needed to activate the channel again (A).  From (4).

A large variety of ion channels in animals are now found to use calmodulin as a subunit and its lobe-specific actions in channel regulation are becoming evident.  See Saimi & Kung (2002) for a review (6).

The Paramecium genome sequencing project (7) has been completed. We tallied 298 clearly recognizable open-reading frames that appear to encode K⁺-channel subunits. It is astonishing  that this single cells have far more K⁺-channel genes than multicellular animals including human.  The most common among the 298 is a class that resembles the CNG/ERG type K⁺-channels A (8) (Fig. 4).  They are presumably functional since 15 such channel genes examined and were found to be transcribed (9).

Fig. 4.  Relatedness of the 15 CNG/ERG-type K+-channel genes examined in detail among the 298 recognizable K+-channel genes recognizes in P. tetraurelia.  From (9).

A massive review on microbial ion channels is available (10).

1.	Haynes et al. (1998) Genetics 149: 947.
2.	Haynes et al. (2000) Genetics 155: 1105.
3.	Kink et al. (1990) Cell  62: 165.
4.	Saimi & Kung (1994) FEBS Let.  350: 155.
5.	Saimi & Ling (1990) Science 249: 1441.
6.	Saimi & Kung (2002) Annu. Rev. Physiol. 64: 289.
7.	Desser et al. (2001) Trends in Genetics 17: 306.
8.	Ling et al. (2001) Genetics  159: 987.
9.	Haynes et al. (2003) Euk. Cell 2: 737.
10.	Martinac et al. (2008) Ion Channels in Microbes. Physio. Reviews 88: 1449.

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