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Summary: Richard Aldrich is interested in the molecular mechanisms of ion channel function and their role in electrical signaling.

Ion channels are the molecular units of electrical signaling in cells. They are proteins that regulate the movement of ions—such as sodium, calcium, and potassium—into and out of cells. They are responsible for the conversion of external sensory signals to the electrical language of the nervous system and for the integration of these signals to generate appropriate behavior. Ion channels are also important for the generation and regulation of the heartbeat, for contraction of muscles, and for the release of hormones in the bloodstream. The body contains a large variety of ion channel types. They are specialized to select for certain species of ions and to selectively open and close in response to a number of different stimuli, such as the binding of a neurotransmitter molecule or a change in the voltage that exists across a cell's membrane.

Our lab is interested in the mechanisms of ion channel function. Recently we have focused on the mechanisms of gating and the physiological roles of voltage- and calcium-activated (BK) potassium channels. The gating of BK channels by both calcium and voltage provides a convenient system to study both ligand- and voltage-dependent gating in the same protein, and to examine their interaction. The broad role of these channels in several tissues provides a rich environment for studying their involvement in cellular and systemic physiological mechanisms. Our recent studies have focused on the urinary bladder.

Interaction Between Voltage and Ca²⁺ in BK Channel Gating
Our biophysical and kinetic measurements of wild-type and mutant BK channel gating have suggested that the voltage sensors and the Ca²⁺-binding sites in each of the four subunits do not interact with each other directly. Frank Horrigan and I determined the Ca²⁺ dependence of voltage sensor movement directly by recording gating currents in the presence and absence of saturating amounts of Ca²⁺. Gating currents are small electrical currents that flow across the cell membrane as the voltage sensors move in response to changes in membrane voltage. We found that the fast gating current associated with voltage sensor activation had only a very small Ca²⁺ dependence, even though Ca²⁺ binding could change the probability of the channel opening greatly. With these observations and others we developed a mechanistic model for BK channel gating that predicts the channels behavior over a wide range of membrane voltage, Ca²⁺ concentration, and open probability. The success of this model suggests strongly that the free-energy contributions to channel activation provided by voltage and by Ca²⁺ binding are simply additive. We concluded that the Ca²⁺-binding sites and the voltage sensors do not interact directly. Rather they both affect channel opening through an allosteric mechanism, by influencing the conformational change between the closed and open conformations. This quantitative model will be valuable in subsequent studies that begin to relate the energetics of channel conformational changes to structural alterations.

Role of the BK β1 Subunit in Urinary Bladder Function
BK channels have diverse physiological properties with tissue-specific distribution. In neurons, BK channels are functionally colocalized with Ca²⁺ channels, shape action potential wave forms, and modulate neurotransmitter release. In smooth muscle, BK channels regulate constriction in arteries, uterine contraction, and filtration rate in the kidney. BK channels are products of a single gene, slowpoke (slo), that encodes the pore-forming α subunit of the channel. In light of the broad tissue localization and diverse functional properties, it is not surprising that a number of mechanisms have been identified that alter slo channel properties. These include alternative splicing of the slo RNA, heteromeric assembly with other subunits, and modification by phosphorylation/dephosphorylation and oxidation/reduction. In addition, accessory β subunits are a means of generating BK channel diversity.

The first β subunit to be identified, β1, is expressed predominantly in smooth muscle and causes dramatic effects, increasing 10-fold the apparent affinity of the channel for Ca²⁺ at 0 mV and greatly shifting the range of voltages over which the channel activates at a given Ca²⁺ concentration. Coassembly of the α and β1 subunits increases the apparent Ca²⁺ sensitivity, slows activation kinetics, and increases charybdotoxin-binding affinity. In smooth muscle, β1-subunit mRNA is enriched and can account for the apparent increased Ca²⁺ sensitivity of BK channels relative to skeletal muscle, where the β1 gene shows little expression.

BK channels composed of the pore-forming α subunit and the smooth muscle–specific β1 subunit play an important role in controlling membrane potential and contractility of urinary bladder smooth muscle. In collaboration with Mark Nelsons laboratory (University of Vermont), Robert Brenner and I used BK channel β1-subunit–knockout mice previously developed in our lab to determine the role of the β1 subunit of the BK channel in controlling the contractions of urinary bladder smooth muscle.

The β-galactosidase reporter (lacZ gene) was targeted to the β1 locus, which provided the opportunity to examine the expression of the β1 subunit in urinary bladder smooth muscle. Based on this approach, the β1 subunit is highly expressed in urinary bladder smooth muscle. The smooth muscle BK channels lacking β1 subunits have reduced activity, consistent with the expected change in BK channel voltage and Ca²⁺ sensitivity mediated by the β1 subunit.

Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of bladder smooth muscle strips from control mice. The effects of the β1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. Strips of bladder smooth muscle from β1-subunit–knockout mice had elevated phasic contraction amplitude and decreased frequency when compared to control strips. Iberiotoxin increased the amplitude and frequency of phasic contractions and smooth muscle tone of bladder strips from the knockout mice, suggesting that BK channels can still regulate contractions in the absence of the β1 subunit. The results indicate that the β1 subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder. These channels are a potential drug target for bladder disorders.

This work was supported in part by the National Institutes of Health.

Last updated January 11, 2006