From funny current to HCN channels: 20 years of excitation.

The "funny" (pacemaker) current has unusual characteristics, including activation on hyperpolarization, permeability to K(+) and Na(+), modulation by internal cAMP, and a tiny, single-channel conductance. In cardiac cells and neurons, pacemaker channels control repetitive activity and excitability. The recent cloning of HCN subunits provides new insight into the molecular basis for the funny channel properties.

The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins.

FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.

Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop.

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor.

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.