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Stephen H Loukin

Publications and source records attributed to Stephen H Loukin.

5 recordsLinked to original sources

Prokaryotic K(+) channels: from crystal structures to diversity.

The deep roots and wide branches of the K(+)-channel family are evident from genome surveys and laboratory experimentation. K(+)-channel genes are widespread and found in nearly all the free-living bacteria, archaea and eukarya. The conservation of basic structures and mechanisms such as the K(+) filter, the gate, and some of the gate's regulatory domains have allowed general insights on animal K(+) channels to be gained from crystal structures of prokaryotic channels. Since microbes are the great majority of life's diversity, it is not surprising that microbial genomes reveal structural motifs beyond those found in animals. There are open-reading frames that encode K(+)-channel subunits with unconventional filter sequences, or regulatory domains of different sizes and numbers not previously known. Parasitic or symbiotic bacteria tend not to have K(+) channels, while those showing lifestyle versatility often have more than one K(+)-channel gene. It is speculated that prokaryotic K(+) channels function to allow adaptation to environmental and metabolic changes, although the actual roles of these channels in prokaryotes are not yet known. Unlike enzymes in basic metabolism, K(+) channel, though evolved early, appear to play more diverse roles than revealed by animal research. Finding and sorting out these roles will be the goal and challenge of the near future.

Archaea↗

The transient receptor potential channel on the yeast vacuole is mechanosensitive.

Ca2+ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca2+ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg = 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca2+ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca2+ concentration and is apparently independent of its known Ca2+ activation, which we now propose to be an amplification mechanism (Ca2+-induced Ca2+ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.

Base Sequence↗

The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K+ channel.

Non-targeted mutagenesis studies of the yeast K(+) channel, TOK1, have led to identification of functional domains common to other cation channels as well as those so far not found in other channels. Among the latter is the ability of the carboxyl tail to prevent channel closure. Here, we show that the tail can fulfill this function in trans. Coexpression of the carboxyl tail with the tail-deleted channel core restores normal channel behavior A Ser/Thr-rich region at its amino end and an acidic stretch at its carboxyl end delineate the minimal region required for tail function. This region of 160 aa apparently forms a discrete functional domain. Interaction of this domain with the channel core is strong, being recalcitrant to removal from excised membrane patches by both high salt and reducing agents. Although the use of a cytoplasmic domain to regulate channel is common among animal channels, by using it as a "foot-in-the-door" to maintain open state appears unique to TOK1, the first fungal K(+) channel studied in depth.

Amino Acid Sequence↗

Carboxyl tail prevents yeast K(+) channel closure: proposal of an integrated model of TOK1 gating.

TOK1 encodes the channel responsible for the prominent outward K(+) current of the yeast plasma membrane. It can dwell in several impermeable states, including a rapidly transiting, K(+)-electromotive-force-dependent "R" (rectifying) state, a voltage-independent "IB" (interburst) state, and a set of [K(+)](ext) and voltage-dependent "C" (closed) states. Whereas evidence suggests that the C states result from the constriction of an inner gate at the cytosolic end of the pore, R is most likely an intrinsic gating property of the K(+) filter. Here, we present evidence that Tok1's carboxyl-tail domain also plays an intimate role in channel gating by dynamically preventing inner-gate closures. We present an integrated model of TOK1 gating in which the filter gate, inner gate, and carboxyl tail interact to produce the various phenomenological states. Both wild-type and tailless behaviors can be replicated using Monte Carlo computer simulations based on this model.

DNA Repair↗