Paul B. Bennett

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K+ channel β-subunits, β2 and β3, which are encoded by distinct genes that map to 3q26.2–27. A single β2 family member and four alternatively spliced variants of β3 were investigated. These subunits have predicted molecular masses of 27.1–31.6 kDa, share ∼30–44% amino acid identity with β1, and exhibit distinct but overlapping expression patterns. Coexpression of the β2 or β3a–c subunits with a BK α-subunit altered the functional properties of the current expressed by the α-subunit alone. The β2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the β3a–c subunits resulted in only partial inactivation of the current, and the β3b subunit conferred an apparent inward rectification. Furthermore, unlike the β1 and β2 subunits, none of the β3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these β-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl-D-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil<MERCK 2<MERCK 1<MERCK 3 with K(D)s 79+/-8, 2.4+/-1.1, 1.3+/-0.9, and approximately 0.16+/-0.02 nM, respectively. The apparent dissociation rate constants among these compounds differed by as much as 394-fold whereas the apparent association constants varied less than 3-fold. Higher affinities were a result of slower drug dissociation kinetics of receptor unbinding. Maximal inhibition was not voltage-dependent and was not statistically different at saturating concentrations by these compounds. These results provide the first detailed functional analysis of the kinetic mechanism of MERCK 1, MERCK 2, and MERCK 3 inhibition of NMDA receptors.

Generation and Characterization of a Cell Line with Inducible Expression of Cav3.2 (T-Type) Channels

Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.

Anchors Aweigh!: Ion Channels, Cytoskeletal Proteins, and Cellular Excitability

Genomics, proteomics, transgenics, molecular medicine: these are some of the scientific catch phrases of the 1990s. The hard work and high expectations of the past decade are beginning to influence reality. Results have accrued to the point that we can begin applying molecular knowledge to therapeutics. Although still in the formative stages, one cannot help but see the vast potential of the exponentially growing molecular knowledge base for understanding physiology and pathophysiology.nnIn recent years, increasing numbers of ion channelopathies—disorders involving mutations in ion channel genes—have been recognized. These disorders include periodic paralyzes, migraine, ataxias, epilepsy, and cardiac arrhythmias to name a few. Research efforts have been directed toward identifying the candidate ion channel genes and their mutations and understanding the functional consequences of these mutations. In many cases, the results have been highly rewarding with a biophysical phenotype that easily correlates with the ultimate clinical phenotype.1 There are also cases where mutations in channel proteins, all of which are known to lead to a clinical disorder, do not have, as yet, a phenotype that is consistent with an interpretable hypothesis. For example, in familial hemiplegic migraine, individual mutations in a Ca2+ channel α subunit gene can apparently cause either loss or gain of function, depending on the mutation.2 3 4 5 6 Perhaps different mutant channels behave differently in their native environment and when interacting with auxiliary proteins. Perhaps additional analysis will reveal a mechanism. Yet at present, it is challenging to reconcile this disparate behavior with the common clinical phenotype of migraine. Presumably, this results from our as-yet limited knowledge …