Assessment Of The Physiological Role Of K~+ Channel OsKAT1.1 In Rice

Potassium channels are key machineries mediating both K⁺ efficient acquisition and redistribution in plant, thus play crucial roles in providing K⁺ nutrition and improving resistances to abiotic stresses. Although K⁺ channels are highly conserved in both structural composition and many classical functional aspects, grown in flooding paddy soil, rice could adapted its K⁺ channels with certain specific function and modes of action to the long-term connection to its growing circumstances. Achievements on K⁺ channels to date are mainly addressed from land-based model plants such as Arabidopsis thaliana and maize etc., our insights to rice K⁺ channels is still limited. Therefore, one of the KAT-typed rice K⁺ channel, OsKAT1.1 was investigated in this thesis. The expression profile and tissue localization of OsKAT1.1 gene was first analyzed to provide pilot guidance for subsequent studies. Functional characteristics and regulatory mechanisms of the channel were studied in detail through electrophysiological approaches, i.e. two-electrode voltage-clamp measurements in Xenopus lavies oocytes and patch-clamp analyses in CHO cells.Major results achieved in the thesis were described as follows:1) Unexpectedly, unlike all KAT-typed K⁺ channel genes which were classically localized to mesophyll cells and guard cells in the leaves, OsKAT1.1 gene was predominantly expressed in roots, where it was up-regulated by physiological drought and down-regulated by salt stress.2) In situ hybridization revealed transcripts of OsKAT1.1 gene were targeted to cortical and central cylinder cells, incorporating a putative function in K⁺ acquisition and translocation in roots.3) Electrophysiological analyses indicated that OsKAT1.1 was a typical low-affinity inward-rectifying K⁺ channel with Km values of 58.2 and 42.5 mmol/L as measured in Xenopus oocytes and CHO cells, respectively.4) Reverse potential (Erev) is a key parameter reflecting the specificity of an ion channel. Ideally, a channel strictly selective to K⁺ ion, would result in a 59mV change in reverse potential (⊿ Erev) in response to 10-fold change in extracellular K⁺ concentration. While the experimental⊿Erev of OsKAT1.1 was 49.9mV, somehow far away from the ideal threshold, implying a weaker specificity to K⁺. 5) This hypothesis was further deduced through pharmacological responses of OsKAT1.1 to well known, specific K⁺ channel blockers. As expected, blockade of TEA to channel current was not voltage dependent, while Ba²⁺ blocked the channel in a voltage dependent manner. Nevertheless, 5mmol/L Ba²⁺ inhibited about 53% and 30% of channel currents at -100 and -140mV, respectively, which were significantly less effective than in known K⁺ channels.6) Besides K⁺, OsKAT1.1 also showed remarkable permeation to NH4+ with a Km to NH4+ of 18.2mmol/L. This constituted a second unique property of OsKAT1.1.7) OsKAT1.1 was a strict voltage-gated channel with a half activation potential (E50) of -126.8mV (pH 7.4), suggesting a role in hyperpolarization-induced K⁺ uptake under K⁺ deficient conditions at root surface. Furthermore, the activity of OsKAT1.1 was stimulated by extracellular acidification. Upon a pH drop from 7.4 to 5.4, the E50 of OsKAT1.1 changed to less negative potential of -90.6mV, with a 2-fold current increase at the same time.8) Genistein, an inhibitor of protein kinase B, when included in the pipet solution used for patch-clamp experiments which thereafter fully diffused into CHO cells, resulted in 40-50% reduction in OsKAT1.1 currents, indicating phosphorylation processes were involved in the functional regulation of OsKAT1.1.The above findings suggest that OsKAT1.1 channel possesses, except certain classical properties as known KAT-typed channels, unique functional characteristics coinciding with efficient K⁺ and NH4+ acquisition in roots. This mechanism may be distinguishably important for the NH4+ -preferential nutrient requirement in rice.